Ca(2+)/calmodulin-mediated regulation of the desensitizing process in G(q) protein-coupled histamine H(1) receptor-mediated Ca(2+) responses in human U373 MG astrocytoma cells

We investigated Ca(2+)/calmodulin (CaM)-mediated regulation of the desensitizing process of the histamine H(1) receptor-mediated increase in intracellular Ca(2+) concentration in human U373 MG astrocytoma cells. The desensitizing process was evaluated by measuring the histamine-induced Ca(2+) responses in cells pretreated with histamine for 15 s-30 min under various conditions. Under normal physiological conditions, desensitization developed with three successive phases : a fast desensitization within 15 s, a transient resensitization at 45 s, and a prompt and sustained redesensitization from 1 to 30 min. Similar processes of desensitization/resensitization occurred even under hypertonic conditions, where histamine-mediated internalization of the histamine H(1) receptor is inhibited. The transient resensitization phase was selectively prevented by deprivation of extracellular Ca(2+) and, even more strikingly, by the presence of W-7 (a CaM antagonist). FK506 and cyclosporin A, Ca(2+)/CaM-dependent protein phosphatase (PP2B) inhibitors, mimicked such effects. In the presence of KN-62, a Ca(2+)/CaM-dependent protein kinase II (CaM kinase II) inhibitor, the early development of desensitization disappeared, allowing a slow and simple development of desensitization. The early processes of desensitization and resensitization were unaffected by W-5, okadaic acid, and KN-04 (less potent inhibitors against CaM, PP2B, and CaM kinase II, respectively) or by GF109203X and chelerythrine (protein kinase C inhibitors). The high-affinity site for histamine was converted to a lower-affinity site by histamine treatment, which also showed a transient restoration phase at 45 s in a manner sensitive to KN-62 and FK506. These results provide the first evidence that Ca(2+)/CaM plays a crucial role in determining the early phase of the desensitizing process via activation of CaM kinase II and PP2B, by regulating agonist affinity for histamine H(1) receptors.     

Na(+) channel regulation by calmodulin kinase II in rat cerebellar granule cells

The effects of specific CaM kinase II inhibitors were investigated on Na(+) channels from rat cerebellar granule cells. A maximal effect of KN-62 was observed at 20 microM and consisted of an 80% reduction of the peak Na(+) current after only a 10-min application. A hyperpolarizing shift of 8 mV in the steady-state inactivation was also observed. KN-04 (20 microM), an inactive analog, had no detectable effect. KN-62 was however inactive on Na(+) currents recorded from Chinese hamster ovary cells expressing the type II A alpha subunit. We have also analyzed the inhibitory effects of CaM kinase II 296-311 and CaM kinase II 281-309 peptides. Both peptides (75 microM) induced a maximum peak Na(+) current reduction within 30 min. Under similar conditions, a truncated peptide CaM kinase II 284-302 was ineffective. These results demonstrate that CaM kinase II acts as a modulator of Na(+) channel activity in cerebellar granule cells.     

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.     

Calcium ion regulates the release of lipase of Fusarium oxysporum.

The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.     

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.     

A selective Ca2+/calmodulin-dependent protein kinase II inhibitor, KN-62, inhibits the enhanced phosphorylation and the activation of tyrosine hydroxylase by 56 mM K+ in rat pheochromocytoma PC12h cells.

Involvement of Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM-kinase II) on the phosphorylation of tyrosine hydroxylase (TH, EC.1.14.16.2) in rat pheochromocytoma, PC12h cells was examined using KN-62, 1-[N,O-Bis(5-isoquinolinsulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipe razine, a selective inhibitor of Ca2+/CaM-kinase II. Both the enhanced phosphorylation of TH and the activated L-3,4-dihydroxyphenylalanine (DOPA) formation in the high K+ depolarization were inhibited by 10 microM KN-62. After incubation of PC12h cells with 10 microM KN-62 for 1 hr, the activation of TH with 3 min incubation of 56 mM K+ was reduced to the basal activity. However, KN-62 did not directly affect the activity of purified rat TH at pH 6.0 or 7.0. These results indicate that Ca2+/CaM-kinase II phosphorylates and activates TH of PC12h cells in the high K+ depolarization.     

Calcium/calmodulin-dependent phosphorylation and activation of human Cdc25-C at the G2/M phase transition in HeLa cells

The human tyrosine phosphatase (p54(cdc25-c)) is activated by phosphorylation at mitosis entry. The phosphorylated p54(cdc25-c) in turn activates the p34-cyclin B protein kinase and triggers mitosis. Although the active p34-cyclin B protein kinase can itself phosphorylate and activate p54(cdc25-c), we have investigated the possibility that other kinases may initially trigger the phosphorylation and activation of p54(cdc25-c). We have examined the effects of the calcium/calmodulin-dependent protein kinase (CaM kinase II) on p54(cdc25-c). Our in vitro experiments show that CaM kinase II can phosphorylate p54(cdc25-c) and increase its phosphatase activity by 2.5-3-fold. Treatment of a synchronous population of HeLa cells with KN-93 (a water-soluble inhibitor of CaM kinase II) or the microinjection of AC3-I (a specific peptide inhibitor of CaM kinase II) results in a cell cycle block in G2 phase. In the KN-93-arrested cells, p54(cdc25-c) is not phosphorylated, p34(cdc2) remains tyrosine phosphorylated, and there is no increase in histone H1 kinase activity. Our data suggest that a calcium-calmodulin-dependent step may be involved in the initial activation of p54(cdc25-c).     

Regulation of endothelial cell barrier function by calcium/calmodulin-dependent protein kinase II

Thrombin-induced endothelial cell barrier dysfunction is tightly linked to Ca(2+)-dependent cytoskeletal protein reorganization. In this study, we found that thrombin increased Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) activities in a Ca(2+)- and time-dependent manner in bovine pulmonary endothelium with maximal activity at 5 min. Pretreatment with KN-93, a specific CaM kinase II inhibitor, attenuated both thrombin-induced increases in monolayer permeability to albumin and decreases in transendothelial electrical resistance (TER). We next explored potential thrombin-induced CaM kinase II cytoskeletal targets and found that thrombin causes translocation and significant phosphorylation of nonmuscle filamin (ABP-280), which was attenuated by KN-93, whereas thrombin-induced myosin light chain phosphorylation was unaffected. Furthermore, a cell-permeable N-myristoylated synthetic filamin peptide (containing the COOH-terminal CaM kinase II phosphorylation site) attenuated both thrombin-induced filamin phosphorylation and decreases in TER. Together, these studies indicate that CaM kinase II activation and filamin phosphorylation may participate in thrombin-induced cytoskeletal reorganization and endothelial barrier dysfunction.     

Evidence that multifunctional calcium/calmodulin-dependent protein kinase II (CaM KII) participates in the meiotic maturation of mouse oocytes

Calcium-dependent signaling pathways are thought to be involved in the regulation of mammalian oocyte meiotic maturation. However, the molecular linkages between the calcium signal and the processes driving meiotic maturation are not clearly defined. The present study was conducted to test the hypothesis that the multi-functional calcium/calmodulin-dependent protein kinase II (CaM KII) functions as one of these key linkers. Mouse oocytes were treated with a pharmacological CaM KII inhibitor, KN-93, or a peptide CaM KII inhibitor, myristoylated AIP, and assessed for the progression of meiosis. Two systems for in vitro oocyte maturation were used: (1) spontaneous gonadotropin-independent maturation and (2) follicle-stimulating hormone (FSH)-induced reversal of hypoxanthine-mediated meiotic arrest. FSH-induced, but not spontaneous germinal vesicle breakdown (GVB) was dose-dependently inhibited by both myristoylated AIP and KN-93, but not its inactive analog, KN-92. However, emission of the first polar body (PB1) was inhibited by myristoylated AIP and KN-93 in both oocyte maturation systems. Oocytes that failed to produce PB1 exhibited normal-appearing metaphase I chromosome congression and spindles indicating that CaM KII inhibitors blocked the metaphase I to anaphase I transition. Similar results were obtained when the oocytes were treated with a calmodulin antagonist, W-7, and matured spontaneously. These results suggest that CaM KII, and hence the calcium signaling pathway, is potentially involved in regulating the meiotic maturation of mouse oocytes. This kinase both participates in gonadotropin-induced resumption of meiosis, as well as promoting the metaphase I to anaphase I transition. Further evidence is therefore, provided of the critical role of calcium-dependent pathways in mammalian oocyte maturation.     

Calmodulin/calmodulin-dependent protein kinase II mediates SAAF-induced motility activation of ascidian sperm

Ca2+-influx and membrane hyperpolarization by sperm-activating and -attracting factor (SAAF) released from the unfertilized egg of the ascidians Ciona cause a transient increase in cAMP, which triggers activation of sperm motility. We demonstrated here the presence of Ca2+-binding protein, calmodulin (CaM), and CaM-dependent kinase II (CaMKII) in the sperm. CaM antagonist, W-7, and CaMKII inhibitor, KN-93, suppressed SAAF-induced membrane hyperpolarization, increase in cAMP, and activation of sperm motility, but inactive analogues of W-7 and KN-93, namely W-5 and KN-92, respectively, did not. Subsequent addition of K+ ionophore, valinomycin, hyperpolarized the plasma membrane, increased cAMP, and conferred motility to the immotile sperm even in the presence of W-7 and KN-93. Addition of IBMX activated motility of sperm, which has been immobilized by W-7 and KN-93. These suggest that increased [Ca2+]i through influx of Ca2+ by SAAF binds to CaM to activate CaMKII. The activated CaMKII may cause membrane hyperpolarization to increase cAMP, which triggers the activation of sperm motility in Ciona.     

CaM kinase II activation and phospholamban phosphorylation by SNP in murine gastric antrum smooth muscles

Elevations in the intracellular Ca(2+) concentration activate the serine/threonine protein kinase Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II). We tested the hypothesis that increased sarco(endo)plasmic reticulum Ca(2+)-ATPase activity by phospholamban (PLB) phosphorylation contributes to smooth muscle relaxation by elevating the sarcoplasmic reticulum (SR) Ca(2+) load and increasing the frequency of Ca(2+) release events from the SR. We have previously shown that caffeine or sodium nitroprusside (SNP) relaxes murine gastric fundus smooth muscles and increases PLB phosphorylation by CaM kinase II. These findings suggest that an increased SR Ca(2+) load increases the frequency of Ca(2+) transients from the SR and results in PLB phosphorylation by CaM kinase II, contributing to caffeine- or SNP-induced relaxation. The aim of the present study was to investigate the effects of SNP on CaM kinase II and PLB phosphorylation in gastric antrum smooth muscles. SNP or 8-bromo-cGMP decreased the basal tone and amplitudes of spontaneous phasic contractions and activated CaM kinase II. SNP-induced relaxation and CaM kinase II activation were blocked by [1,2,4]oxadizolo-[4,3alpha]quinoxaline-1-one (ODQ) and inhibited by cyclopiazonic acid (CPA) or KN-93. SNP also increased PLBSer(16) and PLBThr(17) phosphorylation. Both PLBSer(16) and Thr(17) phosphorylation were ODQ sensitive. However, only PLBThr(17) phosphorylation was inhibited by CPA or KN-93. These results suggest that CaM kinase II activation and PLB phosphorylation participate in the relaxant effect of SNP on murine gastric antrum smooth muscles through a nitric oxide/guanylyl cyclase/cGMP pathway.     

The kinesin-like calmodulin binding protein is differentially involved in cell division

The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end-directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca(2)+ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by approximately 15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated.     

Ca2+/Calmodulin-Dependent Protein Kinase II Inhibitor KN-62 Inhibits Adrenal Medullary Chromaffin Cell Functions Independent of Its Action on the Kinase

Abstract: KN-62, an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced ⁴⁵Ca²⁺ influx, which is essential for functional responses. Carbachol-stimulated ²²Na⁺ influx, veratridine-induced ²²Na⁺ influx, and 56 m M K⁺-evoked ⁴⁵Ca²⁺ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.     

Ischemic preconditioning prevents I/R-induced alterations in SR calcium-calmodulin protein kinase II

Although Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) is known to modulate the function of cardiac sarcoplasmic reticulum (SR) under physiological conditions, the status of SR CaMK II in ischemic preconditioning (IP) of the heart is not known. IP was induced by subjecting the isolated perfused rat hearts to three cycles of brief ischemia-reperfusion (I/R; 5 min ischemia and 5 min reperfusion), whereas the control hearts were perfused for 30 min with oxygenated medium. Sustained I/R in control and IP groups was induced by 30 min of global ischemia followed by 30 min of reperfusion. The left ventricular developed pressure, rate of the left ventricular pressure, as well as SR Ca(2+)-uptake activity and SR Ca(2+)-pump ATPase activity were depressed in the control I/R hearts; these changes were prevented upon subjecting the hearts to IP. The beneficial effects of IP on the I/R-induced changes in contractile activity and SR Ca(2+) pump were lost upon treating the hearts with KN-93, a specific CaMK II inhibitor. IP also prevented the I/R-induced depression in Ca(2+)/calmodulin-dependent SR Ca(2+)-uptake activity and the I/R-induced decrease in the SR CaMK II activity; these effects of IP were blocked by KN-93. The results indicate that IP may prevent the I/R-induced alterations in SR Ca(2+) handling abilities by preserving the SR CaMK II activity, and it is suggested that CaMK II may play a role in mediating the beneficial effects of IP on heart function.     

W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition

Ca(+)-calmodulin (Ca(2+)-CaM)-dependent protein kinase II (Ca(2+)/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca(2+)-occupied CaM, and KN-93, an inhibitor of Ca(2+)/CaMKII, on the K(v)4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC(50) of 92.4 muM. The block was voltage dependent, with an effective electrical distance of 0.18 +/- 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of K(v)4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 muM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated K(v)4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 muM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca(2+)/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca(2+)-CaM, as well as a change in activity of Ca(2+)-CaM-dependent enzymes, including Ca(2+)/CaMKII.     

Developmental distribution of CaM kinase II in the antennal lobe of the sphinx moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

The antennal lobe (primary olfactory center of insects) is completely reorganized during metamorphosis. This reorganization is accompanied by changing patterns of calcium signaling in neurons and glial cells. In the present study, we investigated the developmental distribution of a major calcium-dependent protein, viz., calcium/calmodulin-dependent protein kinase II (CaM kinase II), in the antennal lobe of the sphinx moth Manduca sexta by using a monoclonal antibody. During synaptogenesis (developmental stages 6–10), we found a redistribution of CaM kinase II immunoreactivity, from a homogeneous distribution in the immature neuropil to an accumulation in the neuropil of the glomeruli. CaM kinase II immunoreactivity was less intense in olfactory receptor axons of the antennal nerve and antennal lobe glial cells. Western blot analysis revealed a growing content of CaM kinase II in antennal lobe tissue throughout metamorphosis. Injection of the CaM kinase inhibitor KN-93 into pupae resulted in a reduced number of antennal lobe glial cells migrating into the neuropil to form borders around glomeruli. The results suggest that CaM kinase II is involved in glial cell migration.This work was supported by the DFG LO779/2.     

Xenon induces metaphase arrest in rat astrocytes.

The use of xenon as an almost ideal anesthetic with very little side effects is gaining clinical acceptance, yet its effects on the cellular level are still unclear. It affects intracellular Ca2+-homeostasis but up to now no cellular event or Ca2+-signaling system has been described to be specifically sensitive to xenon. Here we report for the first time a specific effect of xenon on astroglial cells not found with another volatile anesthetic, isoflurane, nor with helium nor with N2: treatment of primary astroglial cells from embryonic rat brain with xenon induces, apart from a slight retardation of the cell cycle, a block at metaphase. Upon removal of xenon cells arrested at metaphase complete their mitosis normally. Even under continuous exposure to xenon, cells can be rescued from metaphase arrest by a small and transient increase in intracellular Ca2+; cells enter anaphase despite the presence of xenon and complete cell division, exhibiting normal rate of chromosome movement and normal chromosome separation. These results suggest that xenon interferes with some Ca2+-dependent regulatory system required for the metaphase-anaphase transition; taking into account its anesthetic effects, xenon may be also involved in the control of glia-mediated signaling transfer.     

Inhibition of insulin secretion by KN-62, a specific inhibitor of the multifunctional Ca2+/calmodulin-dependent protein kinase II.

The effects of KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CamPKII), on insulin secretion and protein phosphorylation were studied in rat pancreatic islets and RINm5F cells. KN-62 was found to dose-dependently inhibit autophosphorylation of CamPKII in subcellular preparations of RINm5F cells (K0.5 = 3.1 +/- 0.3 microM), but had no effect on protein kinase C or myosin light chain kinase activity. KN-62, but not the inactive analogue KN-04, dose-dependently inhibited glucose-induced insulin release (K0.5 = 1.5 +/- 0.5 microM) in a manner similar to the inhibition of CamPKII autophosphorylation. KN-62 (10 microM) inhibited carbachol (in the presence of 8 mM glucose) and potassium-stimulated insulin secretion from islets by 53% and 59%, respectively. These results support a role of CamPKII in glucose-sensitive insulin secretion.     

Ca2+/calmodulin-dependent protein kinase II is an essential mediator in the coordinated regulation of electrocyte Ca2+-ATPase by calmodulin and protein kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.     

Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.