EFFECT OF TRPA1 RECEPTOR ACTIVATION ON TRPV1 CHANNEL DESENSITIZATION IN RAT DORSAL GANGLION NEURONS.

The activity of TRPA1 and TRPV1 channels, their sensitivity to selective activators - allyl isothiocyanate (AITC) and capsaicin (Caps), especially their interaction were studied. The method of microfluorescent microscopy and Ca2+ sensitive dye fura- 2AM. Registration of changes in the concentration of intracellular Ca2+ was performed by using the ratio of fluorescence signals measured at two wavelengths (R = F1/ F2). Researches were conducted on cultured neurons of rat dorsal ganglia (DRG neurons). Application of AITC and Caps on soma of DRG neurons resulted in an increase in intracellular Ca2+. Consistent repeated Caps applications resulted in a significant reduction in the amplitude of Ca2+ transients ( desensitization of TRPV1 channels), which accounted 20,7% of initial value. Further application of selective TRPA1 channel agonist (AITC) resulted in restoration of sensitivity to capsaicin TRPV1 channels ( resensitization TRPV1 channels). Thus, we have established the presence of regulation of TRPV1 channel activity by TRPA1 channels.

[USING THE SERIAL RAMP RECORDINGS FOR RAPID TESTING OF THE GENERATING ABILITY OF IMPULSE ACTIVITY OF ISOLATED HIPPOCAMPAL NEURONS].

In this study we investigated changes of impulse activity of hippocampal neurons of the hippocampus by using ramp recordings. We have described the usage of serial ramp recordings of neuronal electrical activity for rapid testing of the generating ability of isolated hippocampal neurons. An analysis of the data has shown that the proposed protocol of serial ramp recordings allows to define additional characteristics of the neuronal impulse activity: (i) the thresholds for initiation of generation and suppression of the generation, (ii) the shape and amplitude of relationship between the interpulse intervals and neuronal depolarizations. The suggested stimulation protocols and related analysis are tools that can be effectively used to justify influence of chemicals or other experimental factors on the impulse activity of neurons.

Acetylcholine-induced calcium signalling in adrenaline- and noradrenaline-containing adrenal chromaffin cells.

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group (n-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group (m-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on n- and m-type cells, respectively. Cytochemical staining showed that n-cells contained adrenaline, whereas m-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.

Effect of hypoxia on calcium channels depends on extracellular calcium in CA1 hippocampal neurons.

Our previous studies have shown that short lasting hypoxia induces an increase of Ca(2+) influx into the cell through high voltage-activated Ca(2+) channels in hippocampal neurons. This effect was abolished by removing of free Ca(2+) from intracellular solution. The aim of this study was to compare hypoxic responses at different extracellular Ca(2+) concentrations ([Ca(2+)](e)) in hippocampal neurons to ascertain whether the hypoxic sensitivity is restricted to Ca(2+) ions. Whole-cell patch-clamp recordings were made from acutely dissociated CA1 hippocampal neurons of rats. Polarographic method for measurements of O(2) partial pressure was used. Here we found that at 2 mM [Ca(2+)](e) the hypoxic effect was significant (up to approximately 94%), whereas [Ca(2+)](e) elevations to 5 and 15 mM resulted in gradual decreasing of the effect. We found, that total Ca(2+) charge carried into the cell under the hypoxia was similar at all [Ca(2+)](e), whereas Ca(2+) charge carried at normoxia was different for different [Ca(2+)](e), being larger at higher [Ca(2+)](e). These data indicated a saturation of the hypoxic effect due to limitation in the channel conductance. Therefore, we suggested that the hypoxic effect can be connected with increase of channel conductance, and the level of channel conductance at normoxia can determine the amplitude of hypoxic effect.

Intracellular mechanisms of hypoxia-induced calcium increase in rat sensory neurons.

Elevation of cytosolic level of Ca(2+) was measured by spatial screening of freshly isolated dorsal root ganglion neurons loaded with Fura-2AM after subjecting them to a moderate hypoxic solution (pO(2)=10-40 mmHg). Short exposure of neurons to hypoxia resulted in a reversible elevation of intracellular Ca(2+) to about 120% in the cell center and to 80% in the cell periphery. Such elevation could be almost completely eliminated by removal of Ca(2+) or Na(+) from external medium or application of nifedipine, an L-type calcium channel blocker. Remarkable antihypoxic efficiency (58%) was achieved by preapplication of mitochondrial protonophore CCCP. A conclusion is made that in sensory neurons the hypoxia-induced elevation of cytosolic Ca(2+) is induced by combined changes of function in three cell substructures: voltage-operated L-type Ca(2+) and Na(+) channels and Ca(2+) accumulation by mitochondria. Mitochondria are important for spatial difference in the hypoxia-induced Ca(2+) elevation due to their specific location in these neurons.

Scientific and technological aspects of oxygen-sensitive electrodes for measurements of oxygen partial pressure in patch-clamp experiments.

Anoxia/hypoxia induces dramatic changes in brain activity leading to the damages in brain structure. Several minutes of decrease in environmental oxygen partial pressure (P(O2)) can irreversibly destroy nerve cells. Therefore, investigations of intracellular mechanisms responsible for hypoxia-induced changes of mammalian nerve system are very important. On-line adjustment and measurements of P(O2) in bath solution during patch-clamp experiments are especially topical. At the recent time, a special interest on the on-line measurements of oxygen contents in bath solution has appeared as a result of application of old approaches previously used for polarographic measurements of oxygen contents in the blood and tissues. Here we describe the simple method of manufacturing oxygen-sensitive microelectrodes, which can be used with standard patch-clamp amplifier. We also describe the main principles of polarographic method and properties of oxygen-sensitive electrodes used in patch-clamp experiments.

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons.

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.

Selective blockade of N-type calcium channels by levetiracetam.

PURPOSE: We investigated the effect of the new antiepileptic drug (AED) levetiracetam (LEV) on different types of high-voltage-activated (HVA) Ca2+ channels in freshly isolated CA1 hippocampal neurons of rats. METHODS: Patch-clamp recordings of HVA Ca2+ channel activity were obtained from isolated hippocampal CA1 neurons. LEV was applied by gravity flow from a pipette placed near the cell, and solution changes were made by electromicrovalves. Ca2+ channel blockers were used for separation of the channel subtypes. RESULTS: The currents were measured in controls and after application of 1-200 microM LEV. LEV irreversibly inhibited the HVA calcium current by approximately 18% on the average. With a prepulse stimulation protocol, which can eliminate direct inhibition of Ca2+ channels by G proteins, we found that G proteins were not involved in the pathways underlying the LEV inhibitory effect. This suggested that the inhibitory effect arises from a direct action of LEV on the channel molecule. The blocking mechanism of LEV was not related to changes in steady-state activation or inactivation of Ca2+ channels. LEV also did not influence the rundown of the HVA Ca2+ current during experimental protocols lasting approximately 10 min. Finally, LEV at the highest concentration used (200 microM) did not influence the activity of L-, P- or Q-type Ca2+ channels in CA1 neurons, while selectively influencing the activity of N-type calcium channels. The maximal effect on these channels separated from other channel types was approximately 37%. CONCLUSIONS: Our results provide evidence that LEV selectively inhibits N-type Ca2+ channels of CA1 pyramidal hippocampal neurons. These data suggest the existence of a subtype of N-type channels sensitive to LEV, which might be involved in the molecular basis of its antiepileptic action.

Dual action of cytosolic calcium on calcium channel activity during hypoxia in hippocampal neurones.

The role of cytoplasmic calcium level (Ca(i)) in regulation of Ca channel activity during hypoxia was studied in hippocampal neurones from rats. Whole-cell patch clamp recordings in combination with measurements of O(2) partial pressure (pO(2)) were used. Lowering of pO(2) induced a potentiation of HVA Ca channel activity by 25.7% at Ca(i) = 75 nM in comparison with Ca(2+)-free solution. Increase of Ca(i) up to 410 nM slightly increased the effect and significantly slowed the Ca(2+) current run-down. On the other hand, hypoxia increased a steady-state channel inactivation and speeded up the kinetics of Ca(2+) current decay by about 30%. We conclude that moderate hypoxia induces dual action on Ca channels: intracellularly mediated augmentation of Ca influx via Ca channels and their Ca(2+)-dependent inactivation.

Different secretory vesicles can be involved in depolarization-evoked exocytosis.

The relationship between Ca(2+) influx through voltage-activated Ca(2+) channels, resting intracellular Ca(2+) level (Ca(i)) and Ca(2+)-dependent exocytosis was studied in bovine adrenal chromaffin cells by using patch-clamp, capacitance, and fluorescent measurements. It was established that depolarization-induced exocytosis passed over two steps, both of which linearly depend on Ca(i). At Ca(i) lying below critical point (200-300 nM) the slope of the relationship was 4.43 and at Ca(i) exceeding the critical point the slope was equal to 31.63. The vesicular mechanism describing experimental two-step dependence of exocytosis on intracellular Ca(2+) (Ca(i)) is proposed. According to the model at Ca(i) below critical point only small-sized vesicles fuse with plasma membrane, whereas at higher Ca(i), larger vesicles started to fuse.

Electron microscopic evidence for multiple types of secretory vesicles in bovine chromaffin cells.

It has been previously shown that the neuron-like chromaffin cells from the bovine adrenal medulla are heterogeneous. Among other differences, the cells also differed in secretory vesicles represented in their cytoplasm. The present study investigates the types of secretory vesicles in bovine chromaffin cells by electron microscopy. Morphometric analysis revealed five types of electron-dense secretory vesicles in chromaffin cells. These were as follows: elementary large catecholamine-storing chromaffin granules of rounded shape, large dense core vesicles of ovoid and rod-like shapes, small dense core vesicles as well as ribosome-coated vesicles of intermediate density. Among the electron-lucent vesicles there were small synaptic-like microvesicles, endocytotic clathrin-coated vesicles, growth cone vesicles, and emptied large light core vesicles. The structural and functional backgrounds of different types of secretory vesicles are described, focusing on their formation and potential role.

Ultrastructural features of medullary chromaffin cell cultures.

The ultrastructural organization on the fourth day of culture of chromaffin cells isolated from the bovine adrenal medulla was characterized based on electron microscopic and morphological analysis. We established that medullary chromaffin cells could be divided into four morphologically different subtypes. Most cells (49.1% of those examined) had a dense cytoplasm and fine dense granules. Cells with dense cytoplasm and large granules represented a second type of chromaffin cell (21.1%). Cells of the third type had a light cytoplasm, granules with a light halo and a well-developed Golgi apparatus (26.3%). The fourth type of chromaffin cell was characterized by moderately dense cytoplasm with well-expressed varicose rough endoplasmic reticulum (about 3.5%). Among concomitant cell types, cortical adrenal cells from the zona fasciculata and zona glomerulosa, epithelial cells, fibroblasts, lymphocytes, brown lipoblasts and glial Schwann cells were present. Morphological analysis implies that cells with dense cytoplasm and fine granules and those with light cytoplasm and haloed granules (75.4% in total) are adrenaline-containing cells, whereas the cells with dense cytoplasm and large granules (26.3%) contain noradrenaline. Cells with moderately dense cytoplasm and varicose reticulum share common morphological properties with classical glandular cells and, by their properties, were closer to noradrenaline-containing cells. It is concluded that chromaffin cells, which are the main cell type among cultured cells from adult bovine adrenal medulla, are morphologically quite heterogeneous. Other cell types of different nature may also be present in the culture and can locally influence the properties of the investigated medullary chromaffin cells used in electrophysiological experiments.

Different types of calcium channels and secretion from bovine chromaffin cells.

Bovine chromaffin cells possess several types of Ca2+ channels, and influx of Ca2+ is known to trigger secretion. However, discrepant information about the relative importance of the individual subtypes in secretion has been reported. We used whole-cell patch-clamp measurements in isolated cells in culture combined with fura-2 microfluorimetry and pharmacological manipulation to determine the dependence of secretion on different types of Ca2+ channels. We stimulated cells with relatively long depolarizing voltage-clamp pulses in a medium containing 60 mM CaCl2. We found that, within a certain range of pulse parameters, secretion as measured by membrane capacitance changes was mainly determined by the total cumulative charge of Ca2+ inflow and the basal [Ca2+] level preceding a stimulus. Blocking or reducing the contribution of specific types of Ca2+ channels using either 20 microM nifedipine plus 10 microM nimodipine or 1 microM omegaCTxGVIA (omega-conotoxin GVIA) or 2 microM omegaCTxMVIIC (omega-conotoxin MVIIC) reduced secretion in proportion to Ca2+ charge, irrespective of the toxin used. We conclude that for long-duration stimuli, which release a large fraction of the readily releasable pool of vesicles, it is not so important through which type of channels Ca2+ enters the cell. Release is determined by the total amount of Ca2+ entering and by the filling state of the readily releasable pool, which depends on basal [Ca2+] before the stimulus. This result does not preclude that other stimulation patterns may lead to responses in which subtype specificity of Ca2+ channels matters.

High-threshold calcium channel activity in rat hippocampal neurones during hypoxia.

Whole-cell patch clamp recordings in combination with direct control and measurements of O2 tension (pO2) in bath solution were used to determine the sensitivity of Ca2+ channels of cultured hippocampal neurones to hypoxia in glucose free solution. In all tested neurones, a lowering of pO2 to 4/50 mmHg did not induce changes either in magnitude, kinetics or voltage-current relations of total Ca2+ currents, which composed mainly from two types, L-type (64%) and N-type (31%) components. Hypoxia only induced a delay of Ca2+ current run-down about 27.5% and 39% at 50 and 4 mmHg pO2 respectively that presumably depended on changes in cytoplasmic channel-modulatory metabolites. The obtained results demonstrate that Ca2+ channel molecules in cultured hippocampal neurones are themselves insensitive to short-lasting (10-20 min) oxygen and glucose deprivation, and that they are not a principal target for hypoxic influences on hippocampal function.

Calcineurin involvement in the regulation of high-threshold Ca2+ channels in NG108-15 (rodent neuroblastoma x glioma hybrid) cells.

1. We examined the relationship between calcineurin (protein phosphatase 2B (PP2B) and voltage-operated Ca2+ channels (VOCCs) in NG108-15 cells. PP2B expression in NG108-15 cells was altered by transfection with plasmid constructs containing a full length cDNA of human PP2B beta(3) in sense (CN-15) and antisense (CN-21) orientation. 2. Confocal immunocytochemical localization showed that in wild-type cells, PP2B immunoreactivity is uniformly distributed in undifferentiated cells and located at the inner surface of soma membrane and neurites in differentiated cells. 3. To test the Ca2+ dependence of the VOCC, we used high-frequency stimulation (HFS). The L- and N-type VOCCs decreased by 37 and 52%, respectively, whereas the T-type current was only marginally sensitive to this procedure. FK-506 (2 microM), a specific blocker of PP2B, reduced the inhibition of L- and N-type VOCCs induced by HFS by 30 and 33%, respectively. 4. In CN-15-transfected cells overexpressing PP2B, total high-voltage-activated (HVA) VOCCs were suppressed by about 60% at a test potential of +20 mV. Intracellular addition of EGTA or FK-506 into CN-15-transfected cells induced an up to 5-fold increase of HVA VOCCs. 5. These findings indicate that PP2B activity does not influence the expression of HVA Ca2+ channels, but modulates their function by Ca(2+)-dependent dephosphorylation. Thus HVA VOCCs, in a phosphorylated state under control conditions, are downregulated by PP2B upon stimulation, with the major effect on N-type VOCCs.

Diversity and properties of calcium channel types in NG108-15 hybrid cells.

We used an integral of the current-voltage relation as a new evaluation of Ca2+ current component composition in NG108-15 hybrid cells. We determined significant changes in the values and composition of Ca2+ currents during cell differentiation. Only low-voltage-activated Ca2+ currents could be observed in undifferentiated cells; after cell differentiation, high-voltage-activated currents appeared and the total Ca2+ current was increased about 30-fold. By pharmacological and biophysical separation, we determined four main types of Ca2+ channels in differentiated cells: approximately 50%, 20% and 17% of N, T and L types, respectively, and 12% of residual current, which is insensitive to classical blockers of low- and high-voltage-activated currents, with the exception of (omega-conotoxin GVIA. All current components displayed kinetics and pharmacological properties similar to neuronal ones. We also established a significant Ca2+ dependence of omega-conotoxin GVIA to inhibit N-type Ca2+ channels: 10 mM Ca2+ in bath solution reduced the toxin efficacy to block N channels three-fold. The residual component fitted the properties of Q-type Ca2+ channels: it was sensitive to (omega-conotoxin GVIA and very similar to the T-type channel with respect to its kinetics; however, the threshold of its activation was closer to the high-voltage-activated component (- 40 mV). Our results show the functional diversity of Ca2+ channels and demonstrate, for the first time, that presumably the Q type of an alpha1A family, which has biophysical and pharmacological properties distinct from the previously described T, L and N types in these cells, is co-expressed in NG108-15 cells.

Diversity of single potassium channels in isolated snail neurons.

Comparison of K+ channels in mollusk and mammalian neurons has been made to elucidate their fundamental properties. Using patch clamp cell-attached configuration, K+ channels in isolated snail neurons were separated into three subtypes: with big (BKC), medium (MKC) and small (SKC) unitary conductances. BKC and MKC were activated at -30 mV and SKC at more negative potentials. BKC and MKC proved sensitive to TEA, whereas SKC were sensitive to 4-AP. Cd2+ in the pipet decreased unitary conductance of BKC by 55% and of MKC by about 31%. Bath application of 5-HT selectively suppressed MKC. It is suggested that BKC can be referred to large conductance Ca(2+)-dependent K+ currents (KCa), MKC to intermediate conductance KCa and SKC channels comply with the characteristics of A current of mammals. These data show that KCa and A currents may be the most general types of currents generated by K+ channels.

Evidence for colocalization of calcineurin and calcium channels in dorsal root ganglion neurons.

Calcineurin is a serine/threonine protein phosphatase 2B widely distributed in the brain. However, its role in brain function remains unknown. Recent data indicate that calcineurin can participate in long-term depression or long-term potentiation in rat hippocampus. Obviously, calcineurin can also be involved in numerous brain diseases, such as ischaemic hippocampal damage when the protein dephosphorylation system is markedly altered and hyperphosphorylation of the microtubule system in Alzheimer's disease. Besides, abnormal phosphorylation of the cytoskeletal proteins affecting the synaptic signalling can lead to different pathological disorders in the brain. In this study we analysed in more detail the localization of calcineurin in neuronal elements by using confocal microscopy and immunocytochemical approaches to record the enzyme expression in cultured rat dorsal root ganglion neurons. This is the first report showing that calcineurin immunoreactivity is highly expressed in dorsal root ganglion neurons and it is localized mainly near the inner surface of the plasma membrane. Immunostaining of these cells by anti-beta subunits of voltage-operated Ca2+ channels showed that distribution of calcium channel beta-subunit and calcineurin is very similar. Our findings confirm that the function of calcineurin can be directly connected with the activity of voltage-operated calcium channels.

Two distinct receptors operate the cAMP cascade to up-regulate L-type Ca channels.

Previously we have reported that serotonin's (5-hydroxytryptamine or 5-HT) potentiating action on L-type Ca channels is present only in definite neurones from pedal ganglia of the mollusc Helix pomatia [Kostyuk PG, Lu kyanetz EA, Doroshenko PA (1992) Effects of serotonin and cAMP on calcium currents in different neurones of Helix pomatia. Pflügers Arch 420:9-15]. The potentiation is mediated by the cAMP second messenger system and is triggered by 5-HT1-like type receptors. To understand the physiological and pharmacological significance of this phenomenon, we analysed the comparative effects of dopamine (DA) and 5-HT on voltage-operated Ca currents (Ica) in isolated, intracellularly perfused H. pomatia neurones in whole- cell patch-clamp experiments. Two types of effects of DA (1-10 mircoM) and 5-HT (1-10 microM) on Ica were observed in different neurones: reversible inhibition (by about 40% and 20% respectively) or reversible potentiation (up to 65% and 40% respectively) of current amplitude. Neurones insensitive to neurotransmitter application were also observed. Da could induce potentiation of ica only in the same neurones that were similarly sensitive to 5-ht. However, a similar correlation between inhibitory action of neurotransmitters on Ica was not observed. The potentiating effects of 5-HT and DA on Ica were not additive and were mimicked by intracellular cAMP (100 microM) or 20 microgram/ml of the catalytic subunit of protein kinase A. We established that the potentiating effects of neurotransmitters were mediated by two distinct receptors, as the DA receptor antagonist ergometrin (1 microM) selectively inhibited the enhancement of Ica by DA and did not affect the action of 5-HT in the same cell. A similar specificity was observed for the dopaminergic compound, 5-chlortryptamine (10 microM), whereas the classical neuroleptic fluphenazine (10 microM) effectively blocked the 5-HT-evoked effect without significantly changing the action of DA. Methiothepin, an antagonist of 5-HT1 and 5-HT2 receptors, blocked both 5-HT-and DA-evoked effects. The results point out a possible convergence of the two different receptors (5-HT1-like and D1) on the same cAMP-dependent system of phosphorylation in the up-regulation of L-type Ca channel activity in mollusc neurones.

Mechanisms of antagonistic action of internal Ca2+ on serotonin-induced potentiation of Ca2+ currents in Helix neurones.

The influence of internal Ca2+ ions has been investigated during intracellular perfusion of isolated neurones from pedal ganglia of Helix pomatia in which serotonin (5-HT) induces a cyclic-adenosine-monophosphate-(cAMP)-dependent enhancement of high-threshold Ca2+ current (ICa). Internal free Ca2+ ([Ca2+]i) was varied between 0.01 and 10 microM by addition of Ca(2+)-EGTA [ethylenebis(oxonitrilo)tetraacetate] buffer. Elevation of [Ca2+]i depressed the 5-HT effect. The dose/effect curve for the Ca2+ blockade had a biphasic character and could be described by the sum of two Langmuir's isotherms for tetramolecular binding with dissociation constants Kd1 = 0.063 microM and Kd2 = 1 microM. Addition of calmodulin (CM) antagonists (50 microM trifluoperazine or 50 microM chlorpromazine), phosphodiesterase (PDE) antagonists [100 microM isobutylmethylxanthine (IBMX) or 5 mM theophylline] and protein phosphatase antagonists [2 microM okadaic acid (OA)] in the perfusion solution caused "anticalcium" action and modified the Ca2+ binding isotherm. Using the effect of OA and IBMX, two components of the total Ca2+ inhibition were separated and evaluated. In the presence of one of these blockers tetramolecular curves with Kd1 = 0.04 microM and Kd2 = 0.69 microM were obtained describing the activation of the retained unblocked enzyme--PDE or calcineurin (CN) correspondingly. The sum of these isotherms gave a biphasic curve similar to that in control. Leupeptin (100 microM), a blocker of Ca(2+)-dependent proteases did not influence the amplitude of 5-HT effect, indicating that channel proteolysis is not involved in the depression. Our findings show that the molecular mechanism of Ca(2+)-induced suppression of the cAMP-dependent upregulation of Ca2+ channels is due to involvement of two Ca(2+)-CM-dependent enzymes: PDE reducing the cAMP level, and CN causing channel dephosphorylation. No other processes are involved in the investigated phenomenon at a Ca2+ concentration of less than or equal to 10 microM.