Effects of Magnesium Sulfate Administration During Hypoxia on CaM Kinase IV and Protein Tyrosine Kinase Activities in the Cerebral Cortex of Newborn Piglets

The present study tested the hypothesis that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in Ca²⁺/Calmodulin-dependent-kinase (CaM Kinase) IV and Protein Tyrosine Kinase (PTK ) activities. Animals were randomly divided into normoxic (Nx), hypoxic (Hx) and magnesium-pretreated hypoxic (Mg²⁺-Hx) groups. Cerebral hypoxia was confirmed biochemically by measuring ATP and phosphocreatine (PCr) levels. CaM Kinase IV and PTK activities were determined in Nx, Hx and Mg²⁺-Hx newborn piglets. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (270 ± 49), Mg²⁺-Hx (317 ± 82) and Hx (574 ± 41, P < 0.05 vs. Nx and Mg²⁺-Hx) groups. Similarly, there was a significant difference between Protein Tyrosine Kinase activity (pmoles/mg protein/h) in normoxic (378 ± 68), Mg²⁺-Hx (455 ± 67) and Hx (922 ± 66, P < 0.05 vs. Nx and Mg²⁺-Hx ) groups. We conclude that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in CaM Kinase IV and Protein Tyrosine Kinase activities. We propose that by blocking the NMDA receptor ion-channel mediated Ca²⁺-flux, magnesium sulfate administration inhibits the Ca²⁺/calmodulin-dependent activation of CaMKIV and prevents the generation of nitric oxide free radicals and the subsequent increase in PTK activity. As a result, phosphorylation of CREB and Bcl-2 family of proteins is prevented leading to prevention of programmed cell death.     

Mechanism of DNA Fragmentation During Hypoxia in the Cerebral Cortex of Newborn Piglets

We have previously shown that hypoxia results in increased activity of caspase-9, caspase-3 and fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. The present study tested the hypothesis that mechanism of DNA fragmentation during hypoxia in the cerebral cortex of newborn piglets is mediated by caspase-9-dependent caspase-3 activation. Newborn piglets were randomly assigned to normoxic, hypoxic, and hypoxic pretreated with a highly selective caspase-9 inhibitor, Z-LEHD-FMK groups. The data showed that cerebral tissue hypoxia results in increased expression of caspase-activated DNase (CAD) protein in the nucleus and fragmentation of nuclear DNA. A pretreatment with Z-LEHD-FMK attenuated the expression of CAD protein in the nucleus and the fragmentation of nuclear DNA. Based on these results, we conclude that the mechanism by which the nuclear DNA was fragmented is mediated by caspase-9-dependent caspase-3 activation and the consequence of caspase-activated DNase activation in the cerebral cortex of newborn piglets.     

IP3 receptor/Ca2+ channel: from discovery to new signaling concepts

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that induces the release of Ca(2+) from the endoplasmic reticulum (ER). The IP(3) receptor (IP(3)R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP(3)R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP(3)-binding core and N-terminal suppressor sequence of IP(3)R have been identified. An IP(3) indicator (known as IP(3)R-based IP(3) sensor) was developed from the IP(3)-binding core. The IP(3)-binding core's affinity to IP(3) is very similar among the three isoforms of IP(3)R; instead, the N-terminal IP(3) binding suppressor region is responsible for isoform-specific IP(3)-binding affinity tuning. Various pathways for the trafficking of IP(3)R have been identified; for example, the ER forms a meshwork upon which IP(3)R moves by lateral diffusion, and vesicular ER subcompartments containing IP(3)R move rapidly along microtubles using a kinesin motor. Furthermore, IP(3)R mRNA within mRNA granules also moves along microtubules. IP(3)Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP(3)R1 activity. IP(3) has been found to release Ca(2+), but it also releases IRBIT (IP(3)R-binding protein released with IP(3)). IRBIT is a pseudo-ligand for IP(3) that regulates the frequency and amplitude of Ca(2+) oscillations through IP(3)R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP(3)Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP(3)R1, as revealed by cryoelectron microscopy, fits closely with these molecules.     

Relationships of Dopamine, Cortical Oxygen Pressure, and Hydroxyl Radicals in Brain of Newborn Piglets During Hypoxia and Posthypoxic Recovery

Abstract: The present study describes the relationships of extracellular striatal dopamine, cortical oxygen pressure, and striatal hydroxyl radicals in brain of newborn piglets during hypoxia and posthypoxic reoxygenation. Hypoxia was induced by reducing the fraction of inspired oxygen (F_iO₂) from 22% (control) to 7% for 1 h. The F_iO₂ was then returned to the control value and measurements were continued for 2 h. Cerebral oxygen pressure was measured by the oxygen dependent quenching of phosphorescence and extracellular levels of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and hydroxy radicals in the striatum were determined by in vivo microdialysis. Hypoxia decreased the cortical oxygen pressure from 47 ± 2 to 9 ± 1.3 torr (p < 0.001); the levels of extracellular dopamine in the striatum increased to 16,000 ± 3,270% of control (p < 0.01), whereas the levels of DOPAC and HVA decreased to 25.3 ± 6% (p < 0.001) and 36 ± 5% (p < 0.01) of control, respectively. Compared with control, the hydroxyl radical levels at each time point were not significantly increased during hypoxia, although the sum of the measured values was significantly increased (p < 0.05). During the first 5 min after F_iO₂ was returned to 22%, the cortical oxygen pressure increased to control values and stayed at this level for the remainder of the measurement period. The extracellular level of dopamine declined to values not statistically different from control during 40 min of reoxygenation. During the first 10 min of reoxygenation, DOPAC and HVA further decreased and then began to slowly increase. By 70 min of reoxygenation, the values were not significantly different from control. Hydroxyl radicals were above control during the entire period of reoxygenation, with maximal values observed after 100 min of reoxygenation. This increase was largely abolished by injecting the animals with α-methyl-p-tyrosine 5 h before hypoxia, a procedure that depleted the brain of dopamine. Our results suggest that oxidation of striatal dopamine during posthypoxic reoxygenation is at least partly responsible for the observed increase in striatal level of hydroxyl radicals that may exacerbate posthypoxic cerebral injury.     

Effect of Allopurinol on Hypoxia-Induced Modification of the NMDA Receptor in Newborn Piglets

The present study tests the hypothesis that pretreatment with allopurinol, a xanthine oxidase inhibitor, will prevent modification of the NMDA receptor during cerebral hypoxia in newborn piglets. Eighteen newborn piglets were studied. Six normoxic control animals were compared to six untreated hypoxic and six allopurinol (20 mg/kg i.v.) pretreated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 hour and tissue hypoxia was confirmed biochemically by the measurement of ATP and phosphocreatine. Brain cell membrane Na⁺,K⁺-ATPase activity was determined to assess membrane function. Na⁺,K⁺-ATPase activity was decreased from control in both the untreated and treated hypoxic animals (46.0 ± 1.0 vs 37.9 ± 2.5 and 37.3 ± 1.4 mol Pi/mg protein/hr, respectively, p < 0.05). [³H]MK-801 binding was determined as an index of NMDA receptor modification. The receptor density (Bmax) in the untreated hypoxic group was decreased compared to normoxic control (1.09 ± 0.17 vs 0.68 ± 0.22 pmol/mg protein, p < 0.01). The dissociation constant (Kd) was also decreased in the untreated group (10.0 ± 2.0 vs 4.9 ± 1.4 nM, p < 0.01), indicating an increase in receptor affinity. However, in the allopurinol treated hypoxic group, the Bmax (1.27 ± 0.09 pmol/mg protein) was similar to normoxic control and the Kd (8.1 ± 1.2 nM, p < 0.05) was significantly higher than in the untreated hypoxic group. The data show that the administration of allopurinol prior to hypoxia prevents hypoxia-induced modification of the NMDA receptor-ion channel binding characteristics, despite neuronal membrane dysfunction. By preventing NMDA receptor-ion channel modification, allopurinol may produce a neuromodulatory effect during hypoxia and attenuate NMDA receptor mediated excitotoxicity.     

Enhanced ER Ca2+ store filling by overexpression of SERCA2b promotes IP3-evoked puffs

Liberation of Ca²⁺ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP₃R) is modulated by the ER Ca²⁺ content, and overexpression of SERCA2b to accelerate Ca²⁺ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP₃-evoked Ca²⁺ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP₃-evoked puffs to elucidate whether ER [Ca²⁺] may modulate IP₃R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca²⁺ release. SERCA2b and Ca²⁺ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca²⁺ influx and thereby load ER stores. Puffs evoked by photoreleased IP₃ were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca²⁺ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca²⁺ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP₃R gating kinetics.     

Effect of 7-Nitroindazole Sodium on the Cellular Distribution of Neuronal Nitric Oxide Synthase in the Cerebral Cortex of Hypoxic Newborn Piglets

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca⁺⁺-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.     

The Effect of Moderate Hypocapnic Ventilation on Nuclear Ca2+-ATPase Activity, Nuclear Ca2+ Flux, and Ca2+/Calmodulin Kinase IV Activity in the Cerebral Cortex of Newborn Piglets

Previous studies have shown that hypocapnia results in fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. We tested the hypothesis that hypocapnia results in decreased ATP and phosphocreatine (PCr) levels and increased nuclear high-affinity Ca++-ATPase activity, intranuclear Ca++ flux, and CaM kinase IV activity in neuronal nuclei of piglets. Three groups of piglets were ventilated as either hypocapnic (a PaCO2 of 20 mm Hg), normocapnic (a PaCO2 of 40 mm Hg), or corrected hypocapnic (ventilated as hypocapnic but with CO2 added to maintain normocapnia) for 1 h. Tissue ATP levels were lower in the hypocapnic than in the normocapnic group. PCr levels were lower and 45Ca++-influx, Ca++-ATPase activity and CaM kinase IV activity were higher in hypocapnic than in normocapnic or corrected hypocapnic piglets. We conclude that hypocapnia alters nuclear membrane Ca++ flux mechanisms and may alter neuronal phosphorylation mechanisms in the cerebral cortex of piglets.     

Mechanism of Activation of Caspase-9 and Caspase-3 during Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca<Superscript>2+</Superscript>-Influx

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca²⁺–ATPase-dependent increase in nuclear Ca²⁺-influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca²⁺-influx by pretreatment with clonidine, an inhibitor of high affinity Ca²⁺–ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n = 4), hypoxic (Hx, n = 4), and hypoxic treated with clonidine (100 mg/kg) (Hx–Cl, n = 5). Anesthetized, ventilated animals were exposed to an FiO₂ of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (μmoles/g brain) was 4.6 ± 0.3 in Nx, 1.7±0.4 in Hx (P < 0.05 vs. Nx), and 1.5 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). PCr (μmoles/g brain) was 3.6 ± 0.4 in Nx, 1.1 ± 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 ± 0.0642 in Nx and increased to 0.808 ± 0.080 (P < 0.05 vs. Nx and Hx–Cl) in the Hx and 0.562 ± 0.050 in the Hx–Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 ± 1.3 in Nx and 32 ± 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 ± 3.2 in the Hx–Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca²⁺–ATPase-dependent increased nuclear Ca²⁺ during hypoxia results in increased caspase-9 and caspase-3 activity.     

Caspase-3-truncated type 1 inositol 1,4,5-trisphosphate receptor enhances intracellular Ca2+ leak and disturbs Ca2+ signalling

Background information. The IP₃R (inositol 1,4,5‐trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca²⁺ release in virtually all cell types. We have previously demonstrated that caspase‐3‐mediated cleavage of IP₃R1 during cell death generates a C‐terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP₃R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel. Results. In the present study, we demonstrate that expression of the caspase‐3‐cleaved C‐terminus of IP₃R1 increased the rate of thapsigargin‐mediated Ca²⁺ leak and decreased the rate of Ca²⁺ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca²⁺ stores. We detected the truncated IP₃R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP₃R1 blocked sperm factor‐induced Ca²⁺ oscillations and induced an apoptotic phenotype. Conclusions. In the present study, we show that caspase‐3‐mediated truncation of IP₃R1 enhanced the Ca²⁺ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca²⁺ leak is largely compensated by enhanced Ca²⁺‐uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca²⁺ leak acts as a feed‐forward mechanism to divert the cell into apoptosis.     

Effect of Hypoxia on Caspase-3, -8, and -9 Activity and Expression in the Cerebral Cortex of Newborn Piglets

Caspases play an important role in programmed cell death. Caspase-3 is a key executioner of apoptosis, whose activation is mediated by the initiator caspases, caspase-8 and caspase-9. The present study tested the hypothesis that cerebral hypoxia results in increased activation and expression of caspases-3, -8, and -9 in the cytosolic fraction of the cerebral cortex of newborn piglets. To test this hypothesis the activity and expression of caspases-3, -8, and -9 were determined in newborn piglets divided into normoxic and hypoxic groups. Caspase activity was determined spectrofluorometrically using enzyme specific substrates. The expression of caspase protein was assessed by Western blot analysis using enzyme specific antibody. Caspases-3, -8, and -9 activity and expression was significantly higher in the hypoxic group than in the normoxic group. These results demonstrate that hypoxia induces activation and increased expression of both the initiator caspases and the executioner caspase in the cerebral cortex of newborn piglets. We conclude that hypoxia results in stimulation of both the pathways of caspase-3 activation.     

Uncoupled IP3 receptor can function as a Ca2+-leak channel: cell biological and pathological consequences

Ca(2+) release via intracellular release channels, IP(3)Rs (inositol 1,4,5-trisphosphate receptors) and RyRs (ryanodine receptors), is perhaps the most ubiquitous and versatile cellular signalling mechanism, and is involved in a vast number of cellular processes. In addition to this classical release pathway there is limited, but yet persistent, information about less well-defined Ca(2+)-leak pathways that may play an important role in the control of the Ca(2+) load of the endo(sarco)plasmic reticulum. The mechanisms responsible for this 'basal' leak are not known, but recent data suggest that both IP(3)Rs and RyRs may also operate as Ca(2+)-leak channels, particularly in pathological conditions. Proteolytic cleavage or biochemical modification (such as hyperphosphorylation or nitrosylation), for example, occurring during conditions of cell stress or apoptosis, can functionally uncouple the cytoplasmic control domains from the channel domain of the receptor. Highly significant information has been obtained from studies of malfunctioning channels in various disorders; for example, RyRs in cardiac malfunction or genetic muscle diseases and IP(3)Rs in neurodegenerative diseases. In this review we aim to summarize the existing information about functionally uncoupled IP(3)R and RyR channels, and to discuss the concept that those channels can participate in Ca(2+)-leak pathways.     

Effects of Selenium and Topiramate on Cytosolic Ca2+ Influx and Oxidative Stress in Neuronal PC12 Cells

It has been widely suggested that selenium (Se) deficiency play an important role in the pathophysiology of epilepsy. It has been reported that Se provides protection against the neuronal damage in patients and animals with epilepsy by restoring the antioxidant defense mechanism. The neuroprotective effects of topiramate (TPM) have been reported in several studies but the putative mechanism of action remains elusive. We investigated effects of Se and TPM in neuronal PC12 cell by evaluating Ca²⁺ mobilization, lipid peroxidation and antioxidant levels. PC12 cells were divided into eight groups namely control, TPM, Se, H₂O₂, TPM + H₂O₂, Se + H₂O₂, Se + TPM and Se + TPM + H₂O₂. The toxic doses and times of H₂O₂, TPM and Se were determined by cell viability assay which is used to evaluate cell viability. Cells were incubated with 0.01 mM TPM for 5 h and 500 nM Se for 10 h. Then, the cells were exposed to 0.1 mM H₂O₂ for 10 h before analysis. The cells in all groups except control, TPM and Se were exposed to H₂O₂ for 15 min before analysis. Cytosolic Ca²⁺ release and lipid peroxidation levels were higher in H₂O₂ group than in control, Se and TPM combination groups although their levels were decreased by incubation of Se and TPM combination. However, there is no difference on Ca²⁺ release in TPM group. Glutathione peroxidase activity, reduced glutathione and vitamin C levels in the cells were lower in H₂O₂ group than in control, Se and TPM groups although their values were higher in the cells incubated with Se and TPM groups than in H₂O₂ groups. In conclusion, these results indicate that Se induced protective effects on oxidative stress in PC12 cells by modulating cytosolic Ca²⁺ influx and antioxidant levels. TPM modulated also lipid peroxidation and glutathione and vitamin C concentrations in the cell system.     

Effect of N-Methyl-D-Aspartate Receptor Blockade on the Control of Cerebral O2 Supply/Consumption Balance During Hypoxia in Newborn Pigs

Using dizocilpine (MK-801), we tested the hypothesis that N-methyl-D-aspartate (NMDA) receptors are important controllers of cerebral O₂ supply/consumption balance in newborn piglets both during normoxia and hypoxia. Twenty-five 2 to 7-day-old piglets were anesthetized and divided into four groups: (1) Normoxia (n = 6), (2) Normoxia + MK-801 (n = 6), (3) Hypoxia (n = 6), and (4) Hypoxia + MK-801 (n = 7). Regional cerebral blood flow (rCBF) in ml/min/100 g was measured using ¹⁴C-iodoantipyrine, and we determined arterial and venous O₂ saturations by microspectrophotometry, calculating cerebral O₂ consumption (VO₂) in ml O₂/min/100 g in the cortex, hypothalamus and pons. MK-801 did not significantly affect regional VO₂ or rCBF in normoxic piglets. Hypoxia resulted in an increase in local rCBF compared to controls: from 41 ± 6 to 103 ± 18 in the cortex; 34 ± 7 to 101 ± 20 in the hypothalamus; and 45 ± 10 to 95 ± 11 in the pons. Pretreatment with MK-801 abolished this hypoxic flow effect in the cortex (51 ± 2) and hypothalamus (49 ± 5), but not in the pons (91 ± 17). Similar results were observed for VO₂ with control values of 1.9 ± 0.3, 1.6 ± 0.2 and 2.1 ± 0.3 for the cortex, hypothalamus and pons respectively. Hypoxia resulted in an increase in the VO₂ to 3.9 ± 0.4 (cortex), 3.8 ± 0.6 (hypothalamus) and 3.9 ± 0.8 (pons). Pretreatment with MK-801 prior to hypoxia abolished these effects in the cortex (2.1 ± 0.2) and hypothalamus (2.1 ± 0.2), but not in the pons (2.9 ± 0.2). These findings suggest that NMDA receptors may play a role in the control of cerebral metabolism during hypoxia in this immature porcine model.     

IP3 receptors in cell survival and apoptosis: Ca2+ release and beyond

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) serve to discharge Ca²⁺ from ER stores in response to agonist stimulation. The present review summarizes the role of these receptors in models of Ca²⁺-dependent apoptosis. In particular we focus on the regulation of IP₃Rs by caspase-3 cleavage, cytochrome c, anti-apoptotic proteins and Akt kinase. We also address the evidence that some of the effects of IP₃Rs in apoptosis may be independent of their ion-channel function. The role of IP₃Rs in delivering Ca²⁺ to the mitochondria is discussed from the perspective of the factors determining inter-organellar dynamics and the spatial proximity of mitochondria and ER membranes.     

The cyclic nucleotide-activated conductance in olfactory cilia: Effects of cytoplasmic Mg2+ and Ca2+

Summary Olfactory receptor neurons depolarize in response to odorants. This depolarization is mediated by an increase in intracellular cyclic AMP, which directly gates channels in the membranes of the neuronal cilia. Previous evidence suggests that a Ca²⁺ influx during the odorant response may ultimately play a role in terminating the response. One way Ca²⁺ inside the cell could terminate the odorant response would be to directly inhibit the cAMP-gated channels. In this report the effects of cytoplasmic Ca²⁺ and Mg²⁺ on the cAMP-activated current were measured in single olfactory cilia. Near the neuronal resting potential, cytoplasmic Ca²⁺ and Mg²⁺ only slightly reduced the cAMP-activated current. Even at high levels (1.0mm Ca²⁺ or 5.0mm Mg²⁺), the average inhibition was only around 20%. It is therefore unlikely that an influx of divalent cations terminates the odorant response by a direct effect on the cAMP-gated channels.