KATP channel blockers selectively interact with A1-adenosine receptor mediated modulation of acetylcholine release in the rat hippocampus

In this study the role of ATP-sensitive K⁺ channels (K_ATP channels) in the A₁ receptor mediated presynaptic inhibitory modulation of acetylcholine release was investigated in the rat hippocampus. N⁶-Cyclohexyladenosine (CHA), the selective A₁-adenosine receptor agonist, reduced concentration-dependently the stimulation-evoked (2 Hz, 1 ms, 240 shocks) [³H]acetylcholine ([³H]ACh) release, from in vitro superfused hippocampal slices preloaded with [³H]choline, an effect prevented by the selective A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). By themselves, neither K_ATP channel openers, i.e. diazoxide, pinacidil and cromakalim, nor glibenclamide and glipizide, the inhibitors of K_ATP channels, exerted a significant effect on the resting and evoked release of [³H]ACh. Glibenclamide and glipizide (10–100 μM) completely prevented the inhibitory effect of 0.1 μM CHA and shifted the concentration response curve of CHA to the right. 4-Aminopyridine (10–100 μM), the non-selective potassium channel blocker, increased the evoked release of [³H]ACh, but in the presence of 4-aminopyridine, the inhibitory effect of CHA (0.1 μM) still persisted. Oxotremorine, the M₂ muscarinic receptor agonist, decreased the stimulation-evoked release of [³H]ACh, but its effect was not reversed by glibenclamide. 1,3-Diethyl-8-phenylxanthine (DPX), the selective A₁-antagonist, effectively displaced [³H]DPCPX in binding experiments, while in the case of glibenclamide and glipizide, only slight displacement was observed. In summary, our results suggest that K_ATP channels are functionally coupled to A₁ receptors present on cholinergic terminals of the hippocampus, and glibenclamide and glipizide, by interacting with K_ATP channels, relieve this inhibitory neuromodulation.     

Cerebrovascular effects of glibenclamide investigated using high-resolution magnetic resonance imaging in healthy volunteers

Glibenclamide inhibits sulfonylurea receptor (SUR), which regulates several ion channels including SUR1-transient receptor potential melastatin 4 (SUR1-TRPM4) channel and ATP-sensitive potassium (K_ATP) channel. Stroke upregulates SURl-TRPM4 channel, which causes a rapid edema formation and brain swelling. Glibenclamide may antagonize the formation of cerebral edema during stroke. Preclinical studies showed that glibenclamide inhibits K_ATP channel-induced vasodilation without altering the basal vascular tone. The in vivo human cerebrovascular effects of glibenclamide have not previously been investigated.In a randomized, double-blind, placebo-controlled, three-way cross-over study, we used advanced 3 T MRI methods to investigate the effects of glibenclamide and K_ATP channel opener levcromakalim on mean global cerebral blood flow (CBF) and intra- and extracranial artery circumferences in 15 healthy volunteers. Glibenclamide administration did not alter the mean global CBF and the basal vascular tone. Following levcromakalim infusion, we observed a 14% increase of the mean global CBF and an 8% increase of middle cerebral artery (MCA) circumference, and glibenclamide did not attenuate levcromakalim-induced vascular changes. Collectively, the findings demonstrate the vital role of K_ATP channels in cerebrovascular hemodynamic and indicate that glibenclamide does not inhibit the protective effects of K_ATP channel activation during hypoxia and ischemia-induced brain injury.     

Hypotension dilates pial arteries by KATP and Kca channel activation

Hypotension induced pial artery dilation is prostaglandin-dependent in the newborn pig. Prostaglandins, in turn, elicit vasodilation through cGMP and cAMP dependent mechanisms and K⁺ channel activation contributes to cyclic nucleotide induced vasodilation. The present study was designed to characterize the role of ATP sensitive (K_ATP) and calcium sensitive (K_ca) channel activation in hypotension induced pial artery dilation in newborn pigs equipped with a closed cranial window. Glibenclamide and iberiotoxin, K_ATP and K_ca channel antagonists, attenuated hypotension induced dilation (36±1 vs. 14±2% before and after iberiotoxin). Combined administration of these K⁺ channel antagonists eliminated the vascular response. Hypotension induced dilation was associated with elevated cerebrospinal fluid (CSF) cAMP but not cGMP concentration (1023±29 vs. 1566±39 fmol/ml for cAMP). L-NNA, a nitric oxide (NO) synthase inhibitor, and Rp 8-Br cGMPs, a protein kinase G inhibitor, had no effect but Rp 8-Br cAMPs, a protein kinase A inhibitor, attenuated hypotensive dilation (35±1 vs. 16±2% before and after Rp 8-Br cAMPs). Dilation by the cAMP analogue 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) was attenuated by glibenclamide and iberiotoxin (8±1 and 17±1 vs. 4±1 and 9±1% before and after glibenclamide). These data show that both K_ATP and K_ca channel activation contribute to hypotension induced dilation. These data suggest that dilation during hypotension results from the sequential release of prostaglandins and cAMP, which, in turn, activates both the K_ATP and K_ca channel.     

The ‘glibenclamide-shift’ of centrally-acting antinociceptive agents in mice

Morphine-induced antinociception is antagonized by the K⁺-channel blocker glibenclamide (glyburide; Glib), implicating ATP-sensitive (K_ATP) K⁺ channels in the analgesic effect of opioids. The present study examined the generality of this conclusion by measuring the effect of Glib on supraspinal (intracerebroventricular; i.c.v.) antinociception produced by representative μ-opioids and the non-opioids pilocarpine and twoα₂-adrenoceptor agonists (clonidine and tizanidine) using the mouse tail-flick test. Concurrent administration of Glib (40 μg, i.c.v.) produced a significant rightward shift of the dose-response curve of morphine, levorphanol, methadone, pilocarpine, clonidine and tizanidine; a modest, but not statistically significant, rightward shift of the dose-response curves of the μ-selective peptides DAMGO ([d-Ala²,N-Me-Phe⁴, Gly-ol⁵]-enkephalin) and PL017 ([N-Me-Phe³,d-Pro⁴]-morphiceptin); and no shift of the dose-response curves of alfentanil, carfentanil, fentanyl, sufentanil, or β-endorphin. Glib produced aleftward shift of the dose-response curve of etorphine. These data support the involvement of K_ATP-type K⁺ channels in mediation of supraspinal antinociception, differentiate Glib-sensitive and Glib-insensitive opioid agonists, and reveal fundamental differences among antinociceptive agents in the extent of demonstrable utilization of this transduction pathway.     

Electro-acupuncture preconditioning abrogates the elevation of c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brains induced by glibenclamide, an ATP-sensitive potassium channel blocker

This study aimed to clarify the neuroprotective mechanism of electro-acupuncture (EA) preconditioning on hypoxic-ischemic brain injury (HIBI). Using Western blot, the expression of c-fos protein (c-Fos) and c-jun protein (c-Jun) induced by glibenclamide, an ATP-sensitive potassium (K_ATP) channel blocker was examined from cerebral cortical and hippocampal samples in neonatal hypoxic-ischemic rats, with or without EA preconditioning. EA was performed on Hegu (LI4), a well-known acupoint commonly used in Oriental medicine for the treatment of neuronal injury resulting from hypoxia–ischemia (HI). Preconditioned rats were treated with either diazoxide, a K_ATP channel opener, glibenclamide, or sterile saline injected into the left lateral ventricle (i.c.v.), with or without EA administration before HI insult. Interestingly, low c-Fos and c-Jun expressions were found both in diazoxide and EA groups, 24 h after HI. Furthermore, significant differences in relative optical density (ROD) were found between glibenclamide and HI control groups (P≤0.05), as well as between the group administered glibenclamide after EA and the HI control group (P≤0.05). However, the level of c-Fos and c-Jun expression in the group administered glibenclamide after EA was significantly lower than in the glibenclamide group (P≤0.05). The present findings indicate that the effectiveness of EA preconditioning against HIBI may be mediated via the opening of K_ATP channels.     

Tolbutamide attenuates diazoxide-induced aggravation of hypoxic cell injury

ATP-dependent potassium (K_ATP) channels of neurons are closed in the presence of physiological levels of intracellular ATP and open when ATP is depleted during hypoxia or metabolic damage. The present study investigates hypoxic alterations of purine and pyrimidine nucleotide levels supposed to intracellularly modulate K_ATP channels. In addition, the effects of the K_ATP channel activator diazoxide and its antagonist tolbutamide were investigated on ATP, GTP, CTP and UTP levels in slices of the parietal cortex. Hypoxia was evoked by saturation of the medium with 95% N₂–5% CO₂ instead of 95% O₂–5% CO₂ for 5 min. Nucleotide contents were measured by anion-exchange HPLC in neutralized perchloric acid extracts obtained from slices frozen immediately at the end of incubation. Hypoxia per se decreased purine and pyrimidine nucleoside triphosphate contents. Thus, ATP and GTP contents were reduced to 69.9 and 77.6% of the respective normoxic levels. UTP and CTP contents were even more decreased (to 60.9 and 41.6%), probably because the salvage pathway of these pyrimidine nucleotides is less effective than that of the purine nucleotides ATP and GTP. While tolbutamide (300 μM) had no effect on the hypoxia-induced decrease of nucleotides, diazoxide at 300, but not 30 μM aggravated the decline of ATP, UTP and CTP to 51.8, 37.5 and 28.5% of the contents observed at normoxia; GTP levels also showed a tendency to decrease after diazoxide application. Tolbutamide (300 μM) antagonized the effects of diazoxide (300 μM). Nucleoside diphosphate (ADP, GDP and UDP) levels were uniformly increased by hypoxia. There was no hypoxia-induced increase of ADP contents in the presence of tolbutamide (300 μM). The ATP/ADP, GTP/GDP and UTP/UDP ratios uniformly declined at a low pO₂. However, only the ATP/ADP ratio was decreased further by diazoxide (300 μM). The observed alterations in nucleotide contents may be of importance for long- and short-term processes related to acute cerebral hypoxia. Thus, hypoxia-induced alterations of purine and pyrimidine nucleotide levels may influence the open state of K_ATP-channels during the period of reversible hypoxic cerebral injury. Furthermore, alterations during the irreversible period of cerebral injury may also arise, as a consequence of decreased pyrimidine nucleotide contents affecting cell survival via protein and DNA synthesis.     

Mouth breathing increases the pentylenetetrazole-induced seizure threshold in mice: a role for ATP-sensitive potassium channels

Nasal obstruction and consequent mouth breathing have been shown to change the acid–base balance, producing respiratory acidosis. Additionally, there exists a large body of evidence maintaining that acidosis affects the activity of ATP-sensitive potassium (K_ATP) channels, which play a crucial role in the function of the central nervous system (CNS), for example, in modulating seizure threshold. Thus, in the study described here, we examined whether mouth breathing, induced by surgical ligation of nostrils, could affect the seizure threshold induced by pentylenetetrazole in male NMRI mice. Using the selective K_ATP channel opener (diazoxide) and blocker (glibenclamide), we also evaluated the possible role of K_ATP channels in this process. Our data revealed that seizure threshold was increased 6 to 72 hours after nasal obstruction, reaching a peak 48 hours afterward, compared with either control or sham-operated mice (P < 0.01). There was a significant decrease in pH of arterial blood samples and increase in CO₂ partial pressure (PCO₂) during this time. Systemic injection of glibenclamide (1 and 2 mg/kg, ip, daily) significantly prevented the increase in seizure threshold in 48-hour bilaterally nasally obstructed mice, whereas it had no effect on seizure threshold in sham-operated mice. Systemic injection of diazoxide (25 mg/kg, ip, daily) had no effect on seizure threshold in all groups, whereas higher doses (50 and 100 mg/kg, ip, daily) significantly increased seizure threshold in both 48-hour-obstructed and sham-operated mice. The decrease in seizure threshold induced by glibenclamide (2 mg/kg, ip, daily) was prevented by diazoxide (25 mg/kg, ip, daily). These results demonstrate for the first time that mouth breathing, which could result in respiratory acidosis, increases seizure threshold in mice and K_ATP channels may play a role in this effect.     

Arachidonic acid inhibits both K and Ca currents in isolated type I cells of the rat carotid body

Whole-cell patch-clamp recordings were used to investigate the effects of arachidonic acid (AA) on K⁺ and Ca²⁺ channels in isolated rat type I carotid body cells. AA (2–20 μM) produced a concentration-dependent inhibition of both K⁺ currents and Ca²⁺ channel currents. The effects of AA on K⁺ currents were unaffected by indomethacin (5 μM), phenidone (5 μM) or 1-aminobenzotriazole (3 mM), suggesting that AA did not exert its effects via cyclo-oxygenase, lipoxygenase or cytochrome P-450 (cP-450) metabolism. Our results suggest that AA directly and non-selectively inhibits ionic currents in rat type I carotid body cells.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Vasopressin impairs KATP and Kca channel function after brain injury

This study was designed to characterize the role of vasopressin in impaired pial artery dilation to activators of the ATP sensitive K (K_ATP) and calcium sensitive K (K_ca) channel following fluid percussion brain injury (FPI) in newborn pigs equipped with a closed cranial window. Topical vasopressin was coadministered with the K_ATP and K_ca channel agonists cromakalim and NS1619 in a concentration approximating that observed in CSF following FPI. Vasopressin so administered attenuated pial artery dilation to these K⁺ channel activators under conditions of equivalent baseline diameter during non injury conditions (13±1 and 23±1 vs. 4±1 and 10±2% for cromakalim 10⁻⁸, 10⁻⁶ M before and after vasopressin, respectively). Attenuated responses were fully restored when these agonists were coadministered with vasopressin and the vasopressin antagonist [l-(β-mercapto-β,β-cyclopentamethylene propionic acid) 2-(o-methyl)-Tyr-AVP] (MEAVP). Cromakalim and NS1619 induced pial artery dilation was attenuated following FPI and MEAVP preadministration partially prevented such impairment (13±1 and 23±1, sham control; 2±1 and 5±1, FPI; and 9±1 and 15±2%, FPI-MEAVP pretreated for responses to cromakalim 10⁻⁸, 10⁻⁶ M, respectively). These data show that vasopressin blunts K_ATP and K_ca channel mediated cerebrovasodilation. These data suggest that vasopressin contributes to impaired K_ATP and K_ca channel function after brain injury.     

Effects of mammalian brain extracts and chlormadinone acetate on neuronal Na, K-ATPase and electrogenic Na, K-pump activity in vitro

Acid-acetone extracts of brain (from beef and guinea pig) and chlormadinone acetate (CMA) were compared with ouabain for their ability to inhibit the electrogenic Na⁺,K⁺-pump and the Na⁺,K⁺-ATPase of neuronal tissues. The membrane potential of neurones in the paravertebral sympathetic ganglion of the bullfrog was recorded in K⁺-free Ringer's solution by means of the sucrose gap technique. The potassium activated hyperpolarization (K_H⁺), induced by the re-introduction of potassium, was used as an index of electrogenic Na⁺,K⁺-pumping. The K_H⁺ was blocked by 1 μM ouabain. Na⁺,K⁺-ATPase activity was measured in microsomal membrane preparations of frog and beef brain using a continuous spectrophotometric assay. Although ouabain consistently inhibited beef brain Na⁺,K⁺-ATPase (IC₅₀ = 2.2 μM), acid-acetone extracts prepared from guinea pig and beef brain produced only partial inhibition. Neither of the extracts significantly reduced the K_H⁺ of the frog ganglion. CMA inhibited Na⁺,K⁺-ATPase prepared from bullfrog brain and spinal cord with slightly greater potency (IC₅₀ = 4.5 μM) than did ouabain (IC₅₀ = 10 μM). In contrast, electrogenic Na⁺,K⁺-pumping (i.e. the K_H⁺) in the frog ganglion was not affected by this steroid. It is concluded that although both the extracts and CMA inhibited Na⁺,K⁺-ATPase, neither can be considered ouabain-like due to their failure to affect the electrogenic Na⁺,K⁺-pump in situ.     

Maximal densities of μ, δ, and κ receptors are differentially altered by focal cerebral ischaemia in the mouse

Though opioids are known to have neuroprotective properties, little information is available on the functional state of opioidergic receptors following focal cerebral ischaemia. The present study investigated the evolution of the B_max and K_d for [³H]DAMGO, [³H]DADLE, and [³H]U69,593, respectively, for the μ, δ, and κ opioidergic receptors after permanent focal cerebral ischaemia in mice. While the various K_d were unchanged, μ and δ B_max values were precociously decreased in frontoparietal cortices, earlier than κ receptors, reflecting infarct extension with time. The B_max values for μ and δ receptors were also altered in non-infarcted tissues, such as tissues at risk (e.g., temporal auditory cortex) and exofocal (e.g., contralateral and non-infarcted) cortices. These results suggest that, in non-infarcted areas, the observed changes reflect functional modifications to focal ischaemia.     

ATP-sensitive potassium channels contribute to the time-dependent alteration in the pentylenetetrazole-induced seizure threshold in diabetic mice

Although there is evidence that diabetes affects seizure susceptibility, the underlying mechanism has not been completely understood. Several studies also suggest a pivotal role for K_ATP channels in the seizure modulation. The aim of the present study was to evaluate the seizure threshold induced by pentylenetetrazole in diabetic mice at different times (3 days, 1–8 weeks) after induction of diabetes with streptozocin and to examine the possible role of ATP-sensitive potassium (K_ATP) channels in this manner.Our data showed a time-dependent alteration in the threshold in diabetic mice, reaching a peak on week 2 after streptozocin injection and declining significantly afterwards. The seizure threshold in 8-week diabetic mice was even lower than control levels, though the difference was not significant. The K_ATP channel opener cromakalim (0.1–30 μg/kg, i.p.) significantly increased the seizure threshold in control mice. Although the K_ATP channel blocker glibenclamide (0.5, 1 mg/kg) had no effect, it prevented the effects of the potent dose of cromakalim (30 μg/kg) on seizure threshold in control mice. Glibenclamide (1 mg/kg, i.p.) also decreased the seizure threshold in 2-week diabetic mice to the control levels which was blocked by pre-treatment with cromakalim (10 μg/kg, i.p.). Cromakalim (10 μg/kg, i.p.) significantly increased the seizure threshold in 8-week diabetic mice which was inhibited by pre-treatment with glibenclamide (1 mg/kg, i.p.).We demonstrated a time-dependent alteration in the pentylenetetrazole-induced seizure threshold in diabetic mice. This phenomenon might be due to the probable alteration in the K_ATP channel functioning during the diabetic condition.     

Presynaptic effect of 7-OH-DPAT on evoked [H]-acetylcholine release in rat striatal synaptosomes

The objective of the present experiments was to study the presynaptic effect of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT, a D₂-like dopamine receptor agonist) on [³H]-acetylcholine ([³H]-ACh) release induced by potassium (15 mM, 25 mM and 60 mM), potassium channel-blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA and quinine) and veratridine to gain insight into the mechanisms involved in the activation of the D₂ dopamine-receptor subtype located at striatal cholinergic nerve terminals. 7-OH-DPAT (1 μM) inhibited the evoked [³H]-ACh release induced by K⁺ 15 mM in a similar percentage than that obtained during basal conditions (30% and 27%, respectively). Nevertheless, in the presence of 25 mM and 60 mM of K⁺ the inhibitory effect of 7-OH-DPAT was completely abolished. 4-AP (1–100 μM) and TEA (1 and 5 mM) significantly enhanced [³H]-ACh release, showing 69.32%±7.60% (P<0.001) and 52.27%±5.64% (P<0.001), respectively, at the highest concentrations tested. In these conditions, 7-OH-DPAT (1 μM) inhibited the release induced by potassium channel-blockers 25–27%. Quinine (0.1–1 μM) did not alter [³H]-ACh release either in the presence or absence of 7-OH-DPAT. Veratridine 10 μM evoked [³H]-ACh release in the presence of a low-calcium medium, but in such conditions 7-OH-DPAT (1 μM) did not modify the neurotransmitter release in the absence or presence of veratridine. Present data indicate that activation of the presynaptic D₂ dopamine receptor inhibits the [³H]-ACh release by increasing K⁺ conductance, as high K⁺ concentrations abolished the inhibitory control of 7-OH-DPAT on [³H]-ACh release. This effect could be mediated by potassium channels different from those sensitive to 4-AP, TEA and quinine. In addition, the presynaptic D₂ dopamine-receptor activation seems to not involve changes in intracellular Ca²⁺.     

Up-regulation of functional voltage-dependent sodium channels by cyclic AMP-dependent protein kinase in adrenal medulla

Treatment of cultured bovine adrenal chromaffin cells with dbcAMP increased [³H]STX binding with an EC₅₀ of 126 μM and a half-effective time of 12 h; dbcAMP (1 mM × 18 h) raised theB_max approximately 1.5-fold without altering theK_d value. Forskolin (0.1 mM) or IBMX (0.3 mM) also increased [³H]STX binding, while dbcAMP had no effect. Effects of dbcAMP and forskolin were abolished by H-89, an inhibitor of cAMP-dependent protein kinase. Cycloheximide (10 μg/ml) and actinomycin D (10 μg/ml), inhibitors of protein synthesis, nullified the stimulatory effect of dbcAMP, whereas tunicamycin, an inhibitor of protein glycosylation, had no effect. Treatment with dbcAMP augmented veratridine-induced²²Na influx,⁴⁵Ca influx via voltage-dependent Ca channels and catecholamine secretion, while the same treatment did not alter⁴⁵Ca influx and catecholamine secretion caused by high K (a direct activation of voltage-dependent Ca channels) [25]. Na influx via single Na channel calculated from²²Na influx and [³H]STX binding was quantitatively similar between non-treated and dbcAMP-treated cells. Brevetoxin allosterically enhanced veratridine-induced²²Na influx approximately 3-fold in dbcAMP-treated cells as in non-treated cells. These results suggest that cAMP-dependent protein kinase is involved in the modulation of Na channel expression in adrenal medulla.     

Inhibition of M-type K current by linopirdine, a neurotransmitter-release enhancer, in NG108-15 neuronal cells and rat cerebral neurons in culture

The effect of linopirdine, a neurotransmitter-release enhancer, on the M-type K⁺-current, I_K(M), was examined in NGPM1-27 cells, mouse neuroblastoma×rat glioma NG108-15 cells transformed to express m1-muscarinic acetylcholine (ACh) receptors, using the nystatin-perforated patch-recording mode under voltage-clamp conditions. The application of linopirdine induced the inward current associated with an inhibition of I_K(M), which mimics an excitatory part of the ACh-induced responses in NGPM1-27 cells. The affinity of linopirdine for the inhibition of I_K(M) was 24.7 μM in NGPM1-27 cells. In the presence of linopirdine, ACh failed to evoke a further inward current, but ACh still elicited an outward current, thus suggesting that the Ca²⁺-dependent K⁺ current is rather insensitive to linopirdine. Linopirdine also inhibited another voltage-gated potassium current (I_K(V)) at the concentration of 72.3 μM. Finally, the inhibitory effect of linopirdine on I_K(M) was confirmed in pyramidal neurons acutely dissociated from the rat cerebral cortex at 35.8 μM. The results suggest that linopirdine is thus considered to be an inhibitor of some type of K⁺ channels in both NGPM1-27 cells and the rat cerebral neurons.     

Role of cAMP and K channel-dependent mechanisms in piglet hypoxic/ischemic impaired nociceptin/orphanin FQ-induced cerebrovasodilation

This study was designed to determine the role of altered cAMP and K⁺ channel-dependent mechanisms in impaired pial artery dilation to the newly described opioid, nociceptin/orphanin FQ (NOC/oFQ) following hypoxia/ischemia in newborn pigs equipped with a closed cranial window. Recent studies have observed that NOC/oFQ elicits pial dilation via release of cAMP, which, in turn, activates the calcium sensitive (K_ca) and the ATP-dependent K⁺ (K_ATP) channel. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, while hypoxia (10 min) decreased pO₂ to 35±3 mmHg with unchanged pCO₂. Topical NOC/oFQ (10⁻⁸, 10⁻⁶ M) induced vasodilation was attenuated by ischemia/reperfusion (I+R) and reversed to vasoconstriction by hypoxia/ischemia/reperfusion (H+I+R) at 1 h of reperfusion (control, 9±1 and 16±1%; I+R, 3±1 and 6±1%; H+I+R, −7±1 and −12±1%). Such altered dilation returned to control values within 4 h in I+R animals and within 12 h in H+I+R animals. NOC/oFQ dilation was associated with elevated CSF cAMP in control animals but such biochemical changes were attenuated in I+R animals and reversed to decreases in cAMP concentration in H+I+R animals (control, 1037±58 and 1919±209 fmol/ml; I+R, 1068±33 and 1289±30 fmol/ml; H+I+R, 976±36 and 772±27 fmol/ml for absence and presence of NOC/oFQ 10⁻⁶ M, respectively). Topical 8-Bromo cAMP (10⁻⁸, 10⁻⁶ M) pial dilation was unchanged by I+R but blunted by H+I+R (control, 10±1 and 20±1%; I+R, 11±1 and 20±2%; H+I+R, 0±1 and 0±2%). Pituitary adenylate cyclase activating polypeptide and cromakalim, adenylate cyclase and K_ATP channel activators, respectively, elicited dilation that was blunted by both I+R and H+I+R while NS1619, a K_ca channel activator, elicited dilation that was unchanged by I+R but blunted by H+I+R. These data indicate that impaired NOC/oFQ dilation following I+R results form altered adenylate cyclase and K_ATP channel-dependent mechanisms. These data further indicate that impaired NOC/oFQ dilation following H+I+R results not only from altered adenylate cyclase and K_ATP channel but also from altered cAMP and K_ca channel-dependent mechanisms.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

Adenosine activates ATP-sensitive K currents in pericytes of rat retinal microvessels: role of A1 and A2a receptors

In the CNS, contractile pericytes are positioned on the endothelial walls of microvessels where they are thought to play a role in adjusting blood flow to meet local metabolic needs. This function may be particularly important in the retina where pericytes are more numerous than at any other site. Despite the putative importance of pericytes, knowledge of the mechanisms by which vasoactive molecules, such as adenosine, regulate their function is limited. Using the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on microvessels freshly isolated from the adult rat retina, we found that adenosine reversibly activated a hyperpolarizing current in 98% of the sampled pericytes. This adenosine-induced current is likely to be due to the opening of ATP-sensitive potassium (K_ATP) channels since it had a reversal potential near the equilibrium potential for K⁺, was inhibited by the K_ATP channel blocker, glibenclamide, and was mimicked by pinacidil, which is a K_ATP channel opener. Experiments with specific agonists and antagonists indicated that both the high affinity A₁ and the lower affinity A_2a adenosine receptors provided effective pathways for activating K_ATP currents in pericytes recorded under normal metabolic conditions. However, during chemical ischemia, the A₁ receptor pathway rapidly became ineffective. In contrast, activation of A_2a adenosine receptors continued to open K_ATP channels in ischemic pericytes. These results suggest that the regulation of K_ATP channels via A₁ and A_2a receptors allows adenosine to serve over a broad range of metabolic conditions as a vasoactive signal in the retinal microvasculature.     

Ionic currents on type-I cells of the rabbit carotid body measured by voltage-clamp experiments and the effect of hypoxia

Type-I cells (from rabbit embryos) in primary culture were studied in voltage-clamp experiments using the whole cell arrangement of the patch-clamp technique. With a pipette solution containing 130 mM K⁺ and 3 mM Mg-ATP, large outward currents were obtained positive to a threshold of about −30 mV by clamping cells from −50 mV to different test pulses (−80 to 50 mV). Negative to −30 mV, the slope conductance was low (outward rectification). The outward currents were blocked by external Cs⁺ (5 mM) and partially blocked by TEA (5 mM) and Co²⁺ (1 mM). The initial part of the outward currents during depolarizing voltage pulses exhibited a transient Ca²⁺ inward component partially superimposed to a Ca²⁺-dependent outward current. Inward currents were further characterized by replacing K⁺ with Cs⁺ in the intra- and extracellular solution in order to minimize the outward component and by using 1.8 mM Ca²⁺ or 10.8 mM Ba²⁺ as charge carrier. Slow-inactivating inward currents were recorded at test potentials ranging from −50 to 40 mV (holding potential −80 mV). The maximal amplitude, measured at 10 mV in the U-shaped I–V curve, amounted to 247 ± 103pA(n = 3). This inward current was insensitive to 3 μM TTX, but blocked by 1 mM Co²⁺ and partially reduced by 10 μM D600 and 3 μM PN 200-110. In contrast to outward currents, the inward currents exhibited a ‘run-down’ within about 10 min. Lowering the pO₂ from the control of 150 Torr (air-gassed medium) to 28 Torr had no apparent effect on inward currents, but depressed reversibly outward currents by 28%. In conclusion, it is suggested that type-I cells possess voltage-activated K⁺ and Ca²⁺ channels which might be essential for chemoreception in the carotid body.