cAMP-independent decrease of ATP-sensitive K+ channel activity by GLP-1 in rat pancreatic β-cells

Using the patch-clamp method, we studied the mechanism of depolarization of rat pancreatic beta-cells induced by glucagon-like peptide 1 (7-36) amide (GLP-1). GLP-1 caused depolarization in a concentration-dependent manner (0.2-100 nM). Exendin (9-39) amide, a GLP-1 receptor antagonist, prevented the GLP-1-induced depolarization. GLP-1 reduced tolbutamide-sensitive membrane currents evoked by voltage ramps from -90 to -50 mV, recorded in the perforated whole-cell configuration, suggesting that GLP-1 decreased the activity of the ATP-sensitive K+ channel (KATP). This GLP-1 effect was prevented by exendin (9-39) amide. In cells treated with Rp-cAMPS, an inhibitor of the cAMP-dependent protein kinase (PKA), GLP-1 still caused depolarization and reduced the whole-cell membrane current through KATP. Examined in the cell-attached configuration, 20 nM GLP-1, applied out of the patch, had little effect on KATP activity. In the inside-out configuration, the open time probability and the single-channel conductance of KATP in the absence of ATP inside the membrane were unaffected by the presence of 20 nM GLP-1 in the pipette. In both conditions, application of ATP to the inside of the membrane reduced KATP activity. The half-maximal concentrations (ki) of ATP were 11.6 microM without and 5.6 microM with 20 nM GLP-1 in the pipette (P<0.05). The values of the Hill coefficient (h) were 1.03 without and 1.01 with GLP-1. We conclude that GLP-1 reduces KATP activity by elevating the sensitivity of KATP to ATP, resulting in depolarization of pancreatic beta-cells. This GLP-1 action is independent of the cAMP signalling pathway.     

Effects of intracellular pH on ATP-sensitive K+ channels in mouse pancreatic beta-cells.

1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells.     

Role of the cAMP sensor Epac as a determinant of KATP channel ATP sensitivity in human pancreatic beta-cells and rat INS-1 cells

Protein kinase A (PKA)-independent actions of adenosine 3',5'-cyclic monophosphate (cAMP) are mediated by Epac, a cAMP sensor expressed in pancreatic beta-cells. Evidence that Epac might mediate the cAMP-dependent inhibition of beta-cell ATP-sensitive K(+) channels (K(ATP)) was provided by one prior study of human beta-cells and a rat insulin-secreting cell line (INS-1 cells) in which it was demonstrated that an Epac-selective cAMP analogue (ESCA) inhibited a sulphonylurea-sensitive K(+) current measured under conditions of whole-cell recording. Using excised patches of plasma membrane derived from human beta-cells and rat INS-1 cells, we now report that 2'-O-Me-cAMP, an ESCA that activates Epac but not PKA, sensitizes single K(ATP) channels to the inhibitory effect of ATP, thereby reducing channel activity. In the presence of 2'-O-Me-cAMP (50 microM), the dose-response relationship describing ATP-dependent inhibition of K(ATP) channel activity (NP(o)) is left-shifted such that the concentration of ATP producing 50% inhibition (IC(50)) is reduced from 22 microM to 1 microM for human beta-cells, and from 14 microM to 4 microM for rat INS-1 cells. Conversely, when patches are exposed to a fixed concentration of ATP (10 microM), the administration of 2'-O-Me-cAMP inhibits channel activity in a dose-dependent and reversible manner (IC(50) 12 microM for both cell types). A cyclic nucleotide phosphodiesterase-resistant ESCA (Sp-8-pCPT-2'-O-Me-cAMPS) also inhibits K(ATP) channel activity, thereby demonstrating that the inhibitory actions of ESCAs reported here are unlikely to arise as a consequence of their hydrolysis to bioactive derivatives of adenosine. On the basis of such findings it is concluded that there exists in human beta-cells and rat INS-1 cells a novel form of ion channel modulation in which the ATP sensitivity of K(ATP) channels is regulated by Epac.     

Purinergic activation of BK channels in clonal kidney cells (Vero cells)

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP > UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.     

Identification of Calcium-Activated Potassium Channels in Cultured Equine Sweat Gland Epithelial Cells

The patch-clamp recording technique was used to examine the properties of the K+ channels in cultured equine sweat gland epithelial cells. With symmetric K+ solutions (140 mM), a single population of K+ channels was identified with a slope conductance of 187 pS and a reversal potential of around 0 mV. The channel was selective for K+ over Na+. Channel activity was increased by membrane depolarization. A 10-fold increase in [Ca2+]i produced an approximate 60 mV negative shift in the open state probability (Popen)-voltage curve. Externally applied tetraethylammonium ions (TEA+) caused a rapid and flickery block of the channel and reduced the unitary current amplitude. TEA+ bound to the blocking site with stoichiometry of 1:1 and with a dissociation constant (Kd) of 186 +/- 27 microM at +40 mV. A weak voltage dependence of Kd was observed. Iberiotoxin (100 nM) reduced Popen but had no effect on single-channel conductance. Neither glibenclamide (10 microM) nor intracellular adenosine 5'-triphosphate (ATP, 1 mM) altered channel activity. In addition, ATP, when applied extracellularly, transiently activated the channel by increasing Popen. Channel activity was low around the resting membrane potential in the intact epithelia, indicating that these channels might not contribute to the resting K+ conductance. However, the channel could be activated in a regulated manner. The K+ channels may play a role in transepithelial fluid secretion in sweat gland.     

Alteration of the membrane lipid environment by L-palmitoylcarnitine modulates KATP channels in guinea-pig ventricular myocytes

Sarcolemmal adenosine 5'-triphosphate-sensitive K+ channels (K(ATP)) are dramatically up-regulated by a membrane phospholipid, phosphatidyl-inositol-4,5-bisphosphate (PIP2). During ischaemia, L-palmitoylcarnitine (L-PC), a fatty acid metabolite, accumulates in the sarcolemma and deranges the membrane lipid environment. We therefore investigated whether alteration of the membrane lipid environment by L-PC modulates the K(ATP) channel activity in inside-out patches from guinea-pig ventricular myocytes. L-PC (1 microM) inhibited KATP channel activity, without affecting the single channel conductance, through interaction with Kir6.2. L-PC simultaneously enhanced the ATP sensitivity of the channel [concentration for half-maximal inhibition (IC50) fell from 62.0+/-2.7 to 30.3+/-5.5 microM]. In contrast, PIP2 attenuated the ATP sensitivity (IC50 343.6+/-54.4 microM) and restored Ca2+-induced inactivation of KATP channels (94.1+/-13.7% of the control current immediately before the Ca2+-induced inactivation). Pretreatment of the patch membrane with 1 microM L-PC, however, reduced the magnitude of the PIP2-induced recovery to 22.7+/-6.3% of the control (P<0.01 vs. 94.1+/-13.7% in the absence of L-PC). Conversely, after the PIP2-induced recovery, L-PC's inhibitory action was attenuated, but L-PC partly reversed the PIP2-mediated decrease in the ATP sensitivity (IC50 fell from 310+/-19.2 to 93.1+/-9.8 microM). Thus, interaction between L-PC and PIP2 in the plasma membrane appears to regulate K(ATP) channels.     

Met-enkephalin-induced mobilization of intracellular Ca2+ in rat intracardiac ganglion neurones.

The effects of Met-enkephalin on Ca2+-dependent K+ channel activity were investigated using the cell-attached patch recording technique on isolated parasympathetic neurones of rat intracardiac ganglia. Large-conductance, Ca2+-dependent K+ channels (BK(Ca)) were examined as an assay of agonist-induced changes in the intracellular free calcium ion concentration ([Ca2+]i). These BK(Ca) channels had a conductance of approximately 200 pS and were charybdotoxin- and voltage-sensitive. Caffeine (5 mM), used as a control, evoked a large increase in BK(Ca) channel activity, which was inhibited by 10 microM ryanodine. Met-enkephalin (10 microM) evoked a similar increase in BK(Ca) channel activity, which was dependent on the presence of extracellular Ca2+ and inhibited by either ryanodine (10 microM) or naloxone (1 microM). In Fura-2-loaded intracardiac neurones, Met-enkephalin evoked a transient increase in [Ca2+]i. Met-enkephalin-induced mobilization of intracellular Ca+ may play a role in neuronal excitability and firing behaviour in mammalian intracardiac ganglia.     

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.     

Phosphocreatine as a determinant of K(ATP) channel activity in pancreatic beta-cells

The aim of the present study was to test the hypothesis that a creatine kinase/phosphocreatine system is involved in the regulation of K(ATP) channels in pancreatic beta-cells. The phosphocreatine concentration in isolated mouse islets clearly increased in parallel with the ATP/ADP ratio in response to a rise of the glucose concentration from 0.5 mM to 15 mM. The currents through K(ATP) channels expressed in oocytes of Xenopus laevis were inhibited by injection of phosphocreatine or ATP but not by phosphate or creatine alone. In inside-out patches of beta-cell membranes obtained from native beta-cells, phosphocreatine reduced the open probability of single K(ATP) channels in the presence of ADP but not in the absence of the nucleotide. These experiments suggest the existence of a K(ATP) channel-associated creatine kinase that phosphorylates ADP. The creatine kinase inhibitor iodoacetamide suppressed the glucose-induced oscillations of the cytoplasmic Ca(2+) concentration, [Ca(2+)](c). It is concluded that phosphocreatine serves as a shuttle for energy-rich phosphate from the mitochondria to the plasma membrane. The data provide a novel model for signal transduction to K(ATP) channels in pancreatic beta-cells.     

Large-conductance Ca2+-activated potassium channels in secretory neurons.

Large-conductance Ca2+-activated K+ channels (BK) are believed to underlie interburst intervals and contribute to the control of hormone release in several secretory cells. In crustacean neurosecretory cells, Ca2+ entry associated with electrical activity could act as a modulator of membrane K+ conductance. Therefore we studied the contribution of BK channels to the macroscopic outward current in the X-organ of crayfish, and their participation in electrophysiological activity, as well as their sensitivity toward intracellular Ca2+, ATP, and voltage, by using the patch-clamp technique. The BK channels had a conductance of 223 pS and rectified inwardly in symmetrical K+. These channels were highly selective to K+ ions; potassium permeability (PK) value was 2.3 x 10(-13) cm(3) s(-1). The BK channels were sensitive to internal Ca2+ concentration, voltage dependent, and activated by intracellular MgATP. Voltage sensitivity (k) was approximately 13 mV, and the half-activation membrane potentials depended on the internal Ca2+ concentration. Calcium ions (0.3-3 microM) applied to the internal membrane surface caused an enhancement of the channel activity. This activation of BK channels by internal calcium had a KD(0) of 0.22 microM and was probably due to the binding of only one or two Ca2+ ions to the channel. Addition of MgATP (0.01-3 mM) to the internal solution increased steady state-open probability. The dissociation constant for MgATP (KD) was 119 microM, and the Hill coefficient (h) was 0.6, according to the Hill analysis. Ca2+-activated K+ currents recorded from whole cells were suppressed by either adding Cd2+ (0.4 mM) or removing Ca2+ ions from the external solution. TEA (1 mM) or charybdotoxin (100 nM) blocked these currents. Our results showed that both BK and K(ATP) channels are present in the same cell. Even when BK and K(ATP) channels were voltage dependent and modulated by internal Ca2+ and ATP, the profile of sensitivity was quite different for each kind of channel. It is tempting to suggest that BK and KATP channels contribute independently to the regulation of spontaneous discharge patterns in crayfish neurosecretory cells.     

Functional expression of a subtype of the Ca2+ channels in the embryonic rat hippocampus as detected by dual-fluorescence flow cytometric analysis

We have analyzed the expression of the Ca2+ channels in hippocampal cell suspensions from the 18- to 20-day-old rat embryo using dual-fluorescence flow cytometry. A high concentration of K+ induced elevation of the cytoplasmic free Ca2+ concentration ([Ca2+]i) as well as membrane depolarization. The high K+-evoked [Ca2+]i increase was inhibited by phenytoin, but not by either nifedipine or nicardipine. These agents had no effect on the high K+-induced membrane depolarization. These findings suggest that a subtype corresponding to the low voltage-activated Ca2+ channel is expressed in the embryonic rat hippocampal cells.     

Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2+

The effects of various stimulants of insulin release on cytosolic free Ca2+, [Ca2+]i, in dispersed and cultured pancreatic beta-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca2+]i was 158 +/- 7 nM. After increasing glucose to 20 mM there was a lag-period of 1-2 min before [Ca2+]i gradually rose, reaching a new plateau 60% higher after 5-6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca2+]i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the beta-cell function was evident from a marked increase of cytosolic [Ca2+]i after depolarization evoked by high extracellular K+. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca2+]i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca2+]i rather than by increasing the cytosolic concentration of the ion.     

Bursting activity in leech Retzius neurons induced by low external chloride

We have investigated the bursting activity of Retzius neurons in the central nervous system of the leech Hirudo medicinalis as induced in Cl(-)-free saline by measuring membrane potential, membrane current and the intracellular calcium concentration ([Ca2+]i), using fura-2 or Oregon-Green488-Bapta-1. The Retzius neurons changed their low tonic firing to rhythmical bursting activity when the extracellular Cl- concentration ([Cl-]o) was lowered to 1 mM or less. In Cl(-)-free saline (Cl- exchanged by gluconate), bursting was accompanied by a rise in intracellular Ca2+ in both cell body and axon, which oscillated in synchrony with the bursts. The Ca2+ transients depended on the amplitude and duration of the depolarization underlying the burst, and were presumably due to Ca2+ influx through voltage-dependent Ca2+ channels. In Ca(2+)-free, EGTA-buffered saline or in the presence of Ca2+ channel blockers verapamil (1 mM) or diltiazem (500 microM) the depolarizations underlying the bursts in Cl(-)-free saline were enhanced in amplitude and duration. Bursting was not affected by depleting the intracellular Ca2+ stores with cyclopiazonic acid. The depolarization in Cl(-)- and Ca(2+)-free saline did not evoke intracellular Ca2+ changes. The burst-underlying membrane depolarization induced by Cl- removal was found to be due to a Na(+)-dependent persistent inward current and could be inhibited by saxitoxin (25-50 microM). The results suggest that a persistent Na+ current is generated in Cl(-)-free saline and induces the depolarization underlying rhythmic activity, and that presumably Ca(2+)-induced K+ currents modulate the bursting behaviour.     

Properties of a Ca(2+)-activated large conductance K(+) channel with ATP sensitivity in human renal proximal tubule cells

The properties of a native Ca(2+)-activated large conductance K(+) channel (BK channel) present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs) were investigated by using the patch-clamp technique. The slope conductance of the BK channel was about 295 pS, and the channel was selective to K(+) over Na(+), with a selectivity ratio of about 12.2. The activity of the channel was almost maximally enhanced by 10(-4 )M or more Ca(2+) in the cytoplasmic surface of the patch membrane and was markedly diminished by reducing the cytoplasmic Ca(2+) to 10(-6) M at the membrane potential of about 0 mV. The depolarization of the patch membrane also enhanced the channel activity, and hyperpolarization lowered it. K(+) channel blockers, Ba(2+) (0.1-1 mM), tetraethylammonium (1 mM), and charybdotoxin (100 nM), were effective for the suppression of channel activity. A significant feature of the K(+) channel was that channel activity maintained by 10(-5)-10(-4 )M Ca(2+) in inside-out patches was inhibited by the addition of ATP (1-10 mM) to the bath solution. ATPgammaS, and a nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), also had inhibitory effects on channel activity. However, an inhibitor of ATP-sensitive K(+) channels, glibenclamide (0.1 mM), induced no appreciable change in channel activity from both intra- and extracellular sides. These results suggest that besides the common natures of the BK channel family such as regulation by cytoplasmic Ca(2+) and membrane potential, the BK channel in RPTECs is directly inhibited by intracellular ATP independent of phosphorylation processes and sulfonylurea receptor.     

Kinetics of the Ca(2+)-activated K+ channel in rat hippocampal neurons

The kinetics of the large-conductance Ca(2+)-activated K+ channel (235 pS in symmetrical 150 mM K+) were examined in the inside-out mode of the patch clamp technique. The open probability of the channel increased when [Ca2+]i, [Sr2+]i, or [Ba2+]i was increased. The [Ca2+]i-response relation was fitted with a Hill coefficient of 2 and half-maximum concentrations of 185, 80, 14.5, and 5.5 microM at -40, -20, +20, and +40 mV, respectively. The channel was blocked by TEA or Ba2+. The open-time histogram showed a single exponential component and the closed-time histogram showed at least two exponential components at various [Ca2+]i. Increasing [Ca2+]i decreased the time constant of the slow component of the closed-time histogram. Cell-attached patch recording revealed activation of the large-conductance Ca(2+)-activated K+ channel (BK channel) during the action potential. The deactivation time course was consistent with the fast after-hyperpolarization. A minimum model of the channel, close(2)-close(1)-open, where the transition from close(2) to close(1) requires the binding of 2 Ca2+, reconstructed quick activation of the channel if [Ca2+]i of 40 microM was assumed.     

KCNQ/Kv7 channel regulation of hippocampal gamma-frequency firing in the absence of synaptic transmission

Synchronous neuronal firing can be induced in hippocampal slices in the absence of synaptic transmission by lowering extracellular Ca2+ and raising extracellular K+. However, the ionic mechanisms underlying this nonsynaptic synchronous firing are not well understood. In this study we have investigated the role of KCNQ/Kv7 channels in regulating this form of nonsynaptic bursting activity. Incubation of rat hippocampal slices in reduced (<0.2 mM) [Ca2+]o and increased (6.3 mM) [K+]o, blocked synaptic transmission, increased neuronal firing, and led to the development of spontaneous periodic nonsynaptic epileptiform activity. This activity was recorded extracellularly as large (4.7 +/- 1.9 mV) depolarizing envelopes with superimposed high-frequency synchronous population spikes. These intraburst population spikes initially occurred at a high frequency (about 120 Hz), which decayed throughout the burst stabilizing in the gamma-frequency band (30-80 Hz). Further increasing [K+]o resulted in an increase in the interburst frequency without altering the intraburst population spike frequency. Application of retigabine (10 microM), a Kv7 channel modulator, completely abolished the bursts, in an XE-991-sensitive manner. Furthermore, application of the Kv7 channel blockers, linopirdine (10 microM) or XE-991 (10 microM) alone, abolished the gamma frequency, but not the higher-frequency population spike firing observed during low Ca2+/high K+ bursts. These data suggest that Kv7 channels are likely to play a role in the regulation of synchronous population firing activity.     

Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway.

Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.     

Nicotinic receptor-mediated intracellular calcium release in cultured bovine adrenal chromaffin cells

When cultured bovine adrenal chromaffin cells were stimulated by a nicotinic agonist, carbamylcholine (0.3 mM) or 1,1-dimethyl-4-phenylpiperazinium (50 microM), in the Ca2+-free medium containing 0.1 mM ethyleneglycoltetraacetic acid, intracellular free Ca2+ concentration ([Ca2+]i) rose from approximately 90 to 149 nM. High K+ (56 mM) and veratridine (50 microM) had no effect on the [Ca2+]i in Ca2+-free medium. The carbamylcholine-evoked rise in [Ca2+]i was blocked by hexamethonium (0.1 mM) but not by atropine (1 microM). Furthermore, the carbamylcholine-evoked rise in [Ca2+]i was inhibited by an intracellular Ca2+ antagonist, 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (10 microM) but not by a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (20 microM). These results show the existence of intracellular Ca2+ store sites, from which Ca2+ is released upon nicotinic receptor stimulation, in cultured adrenal chromaffin cells.     

Effect of protein kinase C inhibitor (H-7) and calmodulin antagonist (W-7) on pertussis toxin-induced IL-1 production by human adherent monocytes. Comparison with lipopolysaccharide as a stimulator of IL-1 production.

Human adherent monocytes stimulated with 1 microgram/ml pertussis toxin (PT) produced interleukin-1 (IL-1), as measured by thymocyte co-stimulation assay and enzyme-linked immunosorbent assay (ELISA), specific for IL-1 alpha and IL-1 beta. To clarify the role of protein kinase C (PKC) and calmodulin in IL-1 production, we investigated the effects of a PKC inhibitor, H-7, and a calmodulin antagonist, W-7 on PT- and lipopolysaccharide (LPS)-induced IL-1 production by monocytes. Addition of 10 microM and 20 microM H-7 to the culture medium markedly suppressed both PT- and LPS-induced IL-1 production. PT-induced IL-1 production was significantly suppressed by 5 microM and 10 microM W-7. However, LPS-induced IL-1 production was not suppressed by W-7 at the concentrations tested. When monocytes were labelled with Quin 2/AM, IL-1 production by monocytes stimulated with PT and LPS was markedly suppressed. These results indicate that different pathways are involved in the IL-1 production by PT and LPS; both calmodulin- and PKC-dependent processes are necessary for the IL-1 production induced by PT, whereas LPS-induced IL-1 production is dependent on the PKC. Inhibition of IL-1 production by interfering with intracellular Ca2+ trafficking in Quin 2/AM-loaded monocytes may be associated with the inhibition of PKC and calmodulin activity.     

Constitutive activity of inwardly rectifying K+ channel at physiological [Ca]i is mediated by Ca2+/CaMK II pathway in opossum kidney proximal tubule cells

Using patch-clamp technique, we studied the role of the Ca(2+)/calmodulin kinase II (CaMK II)-mediated phosphorylation process on the K(+) channel with an inward conductance of 90 pS in opossum kidney proximal tubule cells (OKPCs). The intracellular Ca(2+) concentration ([Ca](i)) was measured by use of the fluorescent dye fura 2. The following results were obtained: (i) In cell-attached patches, the channel activity was inhibited by a decrease in [Ca](i) induced by perfusion with low Ca(2+) (10(-8) M), La(3+) (100 microM), or EGTA/AM (100 microM) contained in the bath solution. The application of KN-62 (10 microM) or KN-93 (5 microM), inhibitors of CaMK II, also inhibited the channel activity. (ii) The membrane potential measured with nystatin-perforated patches was significantly decreased by the fall in [Ca](i) induced by the perfusion with EGTA- or La(3+)-containing solution. Also, the application of KN-62 (10 microM) or KN-93 (5 microM) to the bath significantly decreased the membrane potential. (iii) In inside-out patches, the channel activity was significantly stimulated by the application of CaMK II (300 pM) at 10(-7) M Ca(2+) in the bath. Furthermore, the application of KN-62 (10 microM) to the bath significantly decreased the channel activity. Our findings show that the constitutive activity of inwardly rectifying K(+) channel at physiological [Ca](i) is mediated by the Ca(2+)/CaMK II pathway in OKPCs.