Calmodulin Protein Kinase II Research Articles

Abstract P3146: Cardiac Stress-induced Arrhythmias And Myocardial Infarction Via Specific Genetically Phosphodeleted Sites In Connexin 43 Channels

Connexin 43 (Cx43) gap junction remodeling is observed in various cardiac pathologies and is linked with arrhythmogenesis, myocardial infarction, and sudden death. Here, we aim to characterize the contribution of Cx43 phosphorylation toward mislocalization at the intercalated disc. For this, we used two genetically phospho-deleted Cx43 knock-in mouse lines with serine residues replaced by alanine in the corresponding sites for phosphorylation via mitogen-activated protein kinase (MAPK) (S255/262/279/282A; Cx43MK4) or Ca 2+/ calmodulin protein kinase II (CaMKII) (S325A/S328A/S330A; Cx43S3A). Ca 2+ signaling, cardiac electrical signals, and arrhythmias were assessed via optical mapping and telemetry in wild type, Cx43MK4, and Cx43S3A mice. Upon basal conditions or isoproterenol stimulation, wild-type animals did not display arrhythmogenic behaviors. However, after isoproterenol treatment, Cx43S3A mice showed increased QTc and deadly arrhythmias compared to wild-type animals. Isoproterenol challenge only increased the number of single PVCs in Cx43MK4 mice when compared to wild-type. Isoproterenol also evoked myocardial infarction in Cx43S3A hearts, which is negligible in Cx43MK4 and wild-type mice. Interestingly, GAP19, a Cx43 hemichannel blocker, reduced lethal arrhythmias and myocardial infarction in Cx43S3A mice. Isoproterenol challenge induced an increase in arrhythmogenic Ca 2+ events in both Cx43S3A and Cx43MK4 hearts compared to wild-type mice. However, Cx43S3A also displayed prolonged Ca 2+ decay transients that were reduced in the presence of Gap19. Our results suggest that the absence of Cx43 phosphorylation by CaMKII leads to lateralization and opening of Cx43 hemichannels, and consequently severe arrhythmias and myocardial infarction. On the other hand, the absence of MAPK phosphorylation promotes mainly a reduction of Cx43 levels at the intercalated disc region that is only associated with mild arrhythmias.

Attenuation of Aging-Induced Vascular Dysfunction by Inhibition of Ca2+/Calmodulin Protein Kinase II (CaMKII) in Isolated Renal and Carotid Rat Arteries

An increased prevalence of cardiovascular problems is recorded due to aging. The importance of aging as a potential risk factor for cardiovascular diseases has been increasing in most of the developing countries since populations continue to become older. Age-associated unusual structural changes that develop in blood vessels have been reported such as stiffening of the vasculature and fibrosis. CaMKII is a protein kinase that is present all-over in the body. In this study, it is suggested that acute inhibition of CaMKII-mediated signaling may be an effective method to prevent agingassociated detrimental changes in isolated blood vessels in SD male rats. The inhibitory effect of CaMKII in aging-induced renal/carotid vascular dysfunction was studied using the selective inhibitor KN-93. Isolated ring segments of the renal or carotid arteries were suspended in organ-bath chambers for measurement of isometric tension. Responses to Phenylephrine (PE), Endothelin-1 (ET-1), carbachol and Sodium Nitroprusside (SNP) were determined by measurement of changes in isometric tension. Results from this study showed abnormal vascular responses to vasoactive agonists in the vessels obtained from aged rats. Vasoconstrictor responses to phenylephrine were potentiated, while vasodilator responses to carbachol were reduced in blood vessels obtained from aged rats, in comparison to responses in young rats. Acute incubation of the blood vessels with KN-93 (10-5 M), an inhibitor of CaMKII, resulted in significant improvement in vascular function to both vasoactive agonists. Our results indicated that activation of CaMKII pathway contributes to aging-induced vascular dysfunction. Therefore, development of novel therapies that inhibit CaMKII pathway may prevent or reduce development of vascular abnormalities induced by aging.

CaMKII activation in early diabetic hearts induces altered sarcoplasmic reticulum-mitochondria signaling

Prediabetic myocardium, induced by fructose-rich diet (FRD), is prone to increased sarcoplasmic reticulum (SR)-Ca2+ leak and arrhythmias due to increased activity of the Ca2+/calmodulin protein kinase II (CaMKII). However, little is known about the role of SR-mitochondria microdomains, mitochondrial structure, and mitochondrial metabolisms. To address this knowledge gap we measured SR-mitochondrial proximity, intracellular Ca2+, and mitochondrial metabolism in wild type (WT) and AC3-I transgenic mice, with myocardial-targeted CaMKII inhibition, fed with control diet (CD) or with FRD. Confocal images showed significantly increased spontaneous Ca2+ release events in FRD vs. CD WT cardiomyocytes. [3H]-Ryanodine binding assay revealed higher [3H]Ry binding in FRD than CD WT hearts. O2 consumption at State 4 and hydrogen peroxide (H2O2) production rate were increased, while respiratory control rate (RCR) and Ca2+ retention capacity (CRC) were decreased in FRD vs. CD WT isolated mitochondria. Transmission Electron Microscopy (TEM) images showed increased proximity at the SR-mitochondria microdomains, associated with increased tethering proteins, Mfn2, Grp75, and VDAC in FRD vs. CD WT. Mitochondria diameter was decrease and roundness and density were increased in FRD vs. CD WT specimens. The fission protein, Drp1 was significantly increased while the fusion protein, Opa1 was unchanged in FRD vs. CD WT hearts. These differences were prevented in AC3-I mice. We conclude that SR-mitochondria microdomains are subject to CaMKII-dependent remodeling, involving SR-Ca2+ leak and mitochondria fission, in prediabetic mice induced by FRD. We speculate that CaMKII hyperactivity induces SR-Ca2+ leak by RyR2 activation which in turn increases mitochondria Ca2+ content due to the enhanced SR-mitochondria tethering, decreasing CRC.

Changed expressions of N-methyl-d-aspartate receptors in the brains of rats and primary neurons exposed to high level of fluoride

Expressions of N-methyl-d-aspartic acid receptors (NMDARs) in the brains of rats and primary neurons exposed to high fluoride were investigated. Sprague-Dawley rats were divided randomly into a fluorosis group (50ppm fluoride in the drinking water for 6 months) and controls (<0.5ppm fluoride) and the offspring from these rats sacrificed on postnatal days 1, 7, 14, 21 and 28. The primary cultured neurons from the hippocampus of neonatal rats were treated with 5 and 50ppm fluoride for 48h. NMDAR subunits at protein or mRNA levels were quantified by Western blotting or real-time PCR. The phosphorylated calmodulin-protein kinase II (CaMKII) was determined by Western blotting, concentration of Ca2+ in neurons by laser confocal microscopy and apoptosis by flow cytometry. In the brains of adult rats and pups as well as in primary neurons exposed to high fluoride, the mRNAs encoding GluN1 and GluN2B subunits and the corresponding proteins were elevated, the GluN3A lowered and the GluN2A unchanged. In addition, the level of phosphor-CaMKII was reduced, and Ca2+ influx and apoptosis enhanced in the brains of rats and cultured neurons exposed to high fluoride. The results indicate that such modifications may involve brain damage induced by chronic fluorosis.

Targeting the CaMKII/ERK Interaction in the Heart Prevents Cardiac Hypertrophy.

AimsActivation of Ca2+/Calmodulin protein kinase II (CaMKII) is an important step in signaling of cardiac hypertrophy. The molecular mechanisms by which CaMKII integrates with other pathways in the heart are incompletely understood. We hypothesize that CaMKII association with extracellular regulated kinase (ERK), promotes cardiac hypertrophy through ERK nuclear localization.Methods and ResultsIn H9C2 cardiomyoblasts, the selective CaMKII peptide inhibitor AntCaNtide, its penetratin conjugated minimal inhibitory sequence analog tat-CN17β, and the MEK/ERK inhibitor UO126 all reduce phenylephrine (PE)-mediated ERK and CaMKII activation and their interaction. Moreover, AntCaNtide or tat-CN17β pretreatment prevented PE induced CaMKII and ERK nuclear accumulation in H9C2s and reduced the hypertrophy responses. To determine the role of CaMKII in cardiac hypertrophy in vivo, spontaneously hypertensive rats were subjected to intramyocardial injections of AntCaNtide or tat-CN17β. Left ventricular hypertrophy was evaluated weekly for 3 weeks by cardiac ultrasounds. We observed that the treatment with CaMKII inhibitors induced similar but significant reduction of cardiac size, left ventricular mass, and thickness of cardiac wall. The treatment with CaMKII inhibitors caused a significant reduction of CaMKII and ERK phosphorylation levels and their nuclear localization in the heart.ConclusionThese results indicate that CaMKII and ERK interact to promote activation in hypertrophy; the inhibition of CaMKII-ERK interaction offers a novel therapeutic approach to limit cardiac hypertrophy.

Cutaneous expression of calcium/calmodulin-dependent protein kinase II in rats with type 1 and type 2 diabetes

Changes in calcium–calmodulin protein kinase II (CaMKII) have been well demonstrated in nervous tissue of diabetic animal models. Skin shares the same ectodermal origin as nervous tissue and it is often affected in diabetic patients. The goal of this study was to analyze expression of CaMKII in rat foot pad 2 weeks and 2 months after induction of diabetes type 1 and 2.Forty-two Sprague-Dawley rats were used. Diabetes mellitus type 1 (DM1) was induced with intraperitoneally (i.p.) injected 55mg/kg of streptozotocin (STZ) and diabetes mellitus type 2 (DM2) with a combination of high-fat diet (HFD) and i.p. injection of low-dose STZ (35mg/kg). Two weeks and two months following diabetes induction rats were sacrificed and skin samples from plantar surface of the both hind paws were removed. Immunohistochemistry was performed for detection of total CaMKII (tCaMKII) and its alpha isoform (pCaMKIIα). For detection of intraepidermal nerve fibers polyclonal antiserum against protein gene product 9.5 (PGP 9.5) was used.The results showed that CaMKII was expressed in the skin of both diabetic models. Total CaMKII was uniformly distributed throughout the epidermis and pCaMKIIα was limited to stratum granulosum. The tCaMKII and pCaMKIIα were not expressed in intraepidermal nerve fibers. Two weeks after induction of diabetes in rats there were no significant differences in expression of tCaMKII and pCaMKIIα between DM1 and DM2 compared to respective controls. In the 2-month experiments, significant increase in epidermal expression of tCaMKII and pCaMKIIα was observed in DM1 animals compared to controls, but not in DM2 animals.This study is the first description of cutaneous CaMKII expression pattern in a diabetic model. CaMKII could play a role in transformation of skin layers and contribute to cutaneous diabetic changes. Further research on physiological role of CaMKII in skin and its role in cutaneous diabetic complications should be undertaken in order to elucidate its function in epidermis.

The essential role of transient receptor potential vanilloid 1 in simvastatin-induced activation of endothelial nitric oxide synthase and angiogenesis.

We investigated the role of transient receptor potential vanilloid receptor type 1 (TRPV1) in simvastatin-mediated activation of endothelial nitric oxide synthase (eNOS) and angiogenesis. Fluo-8 NW assay was for Ca(2+) detection; Griess's assay was for NO bioavailability; Western blotting and immunoprecipitation were for protein phosphorylation and interaction; tube formation and Matrigel plug assay were for angiogenesis. In endothelial cells (ECs), treatment with simvastatin time-dependently increased intracellular level of Ca(2+). Pharmacological inhibition or genetic disruption of TRPV1 abrogated simvastatin-mediated elevation of intracellular Ca(2+) in ECs or TRPV1-transfected HEK293 cells. Loss of TRPV1 function abolished simvastatin-induced NO production and phosphorylation of eNOS and calmodulin protein kinase II (CaMKII) in ECs and in aortas of mice. Inhibition of TRPV1 activation prevented the simvastatin-elicited increase in the formation of TRPV1-Akt-CaMKII-AMPK-eNOS complex. In mice, Matrigel plug assay showed that simvastatin-evoked angiogenesis was abolished by TRPV1 antagonist and genetic ablation of TRPV1. Additionally, our results demonstrated that TRP ankyrin 1 (TRPA1) is the downstream effector in the simvastatin-activated TRPV1-Ca(2+) signalling and in the consequent NO production and angiogenesis as evidence by that re-expression of TRPA1 further augmented simvastatin-elicited Ca(2+) influx in TRPV1-expressed HEK293 cells and ablation of TRPA1 function profoundly inhibited the simvastatin-induced increase in the phosphorylation of eNOS and CaMKII, formation of TRPV1-Akt-CaMKII-AMPK-eNOS complex, NO bioavailability, tube formation and angiogenesis in ECs or mice. Simvastatin-induced Ca(2+) influx may through the activation of TRPV1-TRPA1 signalling, which leads to phosphorylation of CaMKII, increases in the formation of TRPV1-CaMKII-AMPK-eNOS complex, eNOS activation, NO production and, ultimately, angiogenesis in ECs.

Cardiac CD47 Drives Left Ventricular Heart Failure Through Ca 2+ ‐CaMKII‐Regulated Induction of HDAC3

BackgroundLeft ventricular heart failure (LVHF) remains progressive and fatal and is a formidable health problem because ever‐larger numbers of people are diagnosed with this disease. Therapeutics, while relieving symptoms and extending life in some cases, cannot resolve this process and transplant remains the option of last resort for many. Our team has described a widely expressed cell surface receptor (CD47) that is activated by its high‐affinity secreted ligand, thrombospondin 1 (TSP1), in acute injury and chronic disease; however, a role for activated CD47 in LVHF has not previously been proposed.Methods and ResultsIn experimental LVHF TSP1‐CD47 signaling is increased concurrent with up‐regulation of cardiac histone deacetylase 3 (HDAC3). Mice mutated to lack CD47 displayed protection from transverse aortic constriction (TAC)‐driven LVHF with enhanced cardiac function, decreased cellular hypertrophy and fibrosis, decreased maladaptive autophagy, and decreased expression of HDAC3. In cell culture, treatment of cardiac myocyte CD47 with a TSP1‐derived peptide, which binds and activates CD47, increased HDAC3 expression and myocyte hypertrophy in a Ca2+/calmodulin protein kinase II (CaMKII)‐dependent manner. Conversely, antibody blocking of CD47 activation, or pharmacologic inhibition of CaMKII, suppressed HDAC3 expression, decreased myocyte hypertrophy, and mitigated established LVHF. Downstream gene suppression of HDAC3 mimicked the protective effects of CD47 blockade and decreased hypertrophy in myocytes and mitigated LVHF in animals.ConclusionsThese data identify a proximate role for the TSP1‐CD47 axis in promoting LVHF by CaKMII‐mediated up‐regulation of HDAC3 and suggest novel therapeutic opportunities.

Sustained Epac activation induces calmodulin dependent positive inotropic effect in adult cardiomyocytes

Cardiac actions of Epac (exchange protein directly activated by cAMP) are not completely elucidated. Epac induces cardiomyocytes hypertrophy, Ca2+/calmodulin protein kinase II (CaMKII) and excitation–transcription coupling in rat cardiac myocytes. Here we aimed to elucidate the pathway cascade involved in Epac sustained actions, as during the initiation of hypertrophy development, where β-adrenergic signaling is chronically stimulated. Rats were treated with the Epac selective activator 8-pCPT during 4weeks and Ca2+ signaling was analyzed in isolated cardiac myocytes by confocal microscopy. We observed a positive inotropic effect manifested by increased [Ca2+]i transient amplitudes. In order to further analyze these actions, we incubated adult cardiomyocytes in the presence of 8-pCPT. The effects were similar to those obtained in-vivo and are blunted by Epac1 knock down. Interestingly, the increase in [Ca2+] transients was abolished by protein synthesis blockade or when the downstream effectors of calmodulin (CaMKII or calcineurin) were inhibited, pointing to calmodulin (CaM) as an important downstream protein in Epac sustained actions. In fact, CaM expression was enhanced by 8-pCPT treatment in isolated cells, as found by Western blots. Moreover, the 8-pCPT-induced, PKA-independent, positive inotropic effect was favored by enhanced extracellular Ca2+ influx via L-type Ca2+ channels. However, 8-pCPT also induced aberrant Ca2+ release as Ca2+ waves and extra [Ca2+]i transients, suggesting proarrhythmic effect. These results provide new insights regarding Epac cardiac actions, suggesting an important role in the initial compensation of the heart to pathological stimuli during the initiation of cardiac hypertrophy, favoring contraction but also arrhythmia risk.

Identification of CaMKII phosphorylation sites in Connexin43 by high-resolution mass spectrometry.

Connexin43 (Cx43) is a major cardiac gap junction channel protein required for normal electrical and contractile activity. Gap junction channel assembly, function, and turnover are regulated by phosphorylation under both normal and disease conditions. The carboxyl terminus (CT) of Cx43 contains numerous amino acid residues that are phosphorylated by protein kinases. However, our knowledge of the specific residues and kinases involved is incomplete. The objective of this study was to identify amino acid residues in the Cx43-CT that are targets of the multifunctional protein kinase, Ca(2+)/calmodulin protein kinase II (CaMKII), an enzyme known to play critical roles in Ca(2+) homeostasis, transcription, apoptosis, and ischemic heart disease. We subjected fusion protein containing the Cx43-CT to phosphorylation by CaMKII in vitro, digestion with Lys-C and trypsin followed by enrichment for phosphorylated peptides using TiO(2), and analysis in an LTQ XL Orbitrap with collision-induced dissociation and electron transfer dissociation. We deduced the sites of modification by interpreting tandem spectra from these "orthogonal" methods of gas phase peptide fragmentation. We have identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts.

Highlights From The Literature

Highlights From The Literature

Ca2+/calmodulin-dependent protein kinase II potentiates ATP responses by promoting trafficking of P2X receptors.

To elucidate the functional link between Ca(2+)/calmodulin protein kinase II (CaMKII) and P2X receptor activation, we studied the effects of electrical stimulation, such as occurs in injurious conditions, on P2X receptor-mediated ATP responses in primary sensory dorsal root ganglion neurons. We found that endogenously active CaMKII up-regulates basal P2X3 receptor activity in dorsal root ganglion neurons. Electrical stimulation causes prolonged increases in ATP currents that lasts up to approximately 45 min. In addition, the total and phosphorylated CaMKII are also up-regulated. The enhancement of ATP currents depends on Ca(2+) and calmodulin and is completely blocked by the CaMKII inhibitor, 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine). Western analyses indicate that electrical stimulation enhances the expression of P2X3 receptors in the membrane and that the enhancement is blocked by the inhibitor. These results suggest that CaMKII up-regulated by electrical stimulation enhances ATP responses by promoting trafficking of P2X receptors to the membrane and may play a key role in the sensitization of P2X receptors under injurious conditions.

A large sustained Ca2+ elevation occurs in unstimulated spines during the LTP pairing protocol but does not change synaptic strength.

Synapses in the CA1 region of the hippocampus undergo bidirectional synaptic modification in response to different patterns of activity. Postsynaptic Ca2+ elevation can trigger either synaptic strengthening or weakening, depending on the properties of the local Ca2+ signal. During the pairing protocol for long-term potentiation (LTP) induction, the cell is depolarized under voltage-clamp and is given low-frequency synaptic stimulation. As an initial step toward understanding the Ca2+ dynamics during this process, we used confocal microscopy to study the Ca2+ signals in spines evoked by the depolarization itself. This depolarization activates voltage-dependent Ca2+ channels (VDCC), but whether these channels inactivate rapidly or remain functional throughout the long depolarizations used in the pairing protocol remains unknown. Cells were depolarized to 0 mV for 2-3 min. This depolarization led to a large initial elevation of Ca2+ in spines that never decayed back to resting levels. The maintained signal was close to the Kd of the low-affinity (5 microM) Ca2+ dye, Magnesium Green. We attempted to determine the functional role of this elevation, using the Ca2+-channel blocker D-890. The addition of D-890 in the internal solution produced a nearly complete abolition of the Ca2+ elevation during depolarization. Under these conditions, the NMDA conductance was normal, but LTP was almost completely blocked. This might suggest the importance of VDCC in LTP; however, we found that high concentrations of D-890 can directly inhibit calmodulin protein kinase II (CaMKII), an enzyme required for LTP induction. Thus, whereas D-890 is a useful tool for blocking postsynaptic VDCC, it cannot be used to study the contribution of these channels to plasticity. We conclude that the activation of VDCC produces a large and persistent elevation of Ca2+ in all spines, but does not produce either LTP or long-term depression (LTD) in the absence of synaptic stimulation. The possible reasons for this are discussed.

Calcium/calmodulin-dependent protein kinase II regulates Caenorhabditis elegans locomotion in concert with a G(o)/G(q) signaling network.

Caenorhabditis elegans locomotion is a complex behavior generated by a defined set of motor neurons and interneurons. Genetic analysis shows that UNC-43, the C. elegans Ca(2+)/calmodulin protein kinase II (CaMKII), controls locomotion rate. Elevated UNC-43 activity, from a gain-of-function mutation, causes severely lethargic locomotion, presumably by inappropriate phosphorylation of targets. In a genetic screen for suppressors of this phenotype, we identified multiple alleles of four genes in a G(o)/G(q) G-protein signaling network, which has been shown to regulate synaptic activity via diacylglycerol. Mutations in goa-1, dgk-1, eat-16, or eat-11 strongly or completely suppressed unc-43(gf) lethargy, but affected other mutants with reduced locomotion only weakly. We conclude that CaMKII and G(o)/G(q) pathways act in concert to regulate synaptic activity, perhaps through a direct interaction between CaMKII and G(o).

Short- and Long-Term Memory Are Differentially Affected by Metabolic Inhibitors Given into Hippocampus and Entorhinal Cortex

Rats were implanted with cannulae in the CA1 area of the dorsal hippocampus or in the entorhinal cortex and trained in one-trial step-down inhibitory avoidance. Two retention tests were carried out in each animal, one at 1.5 h to measure short-term memory (STM) and another at 24 h to measure long-term memory (LTM). The purpose of the present study was to screen the effect on STM of various drugs previously shown to affect LTM of this task when given posttraining at the same doses that were used here. The drugs and doses were the guanylyl cyclase inhibitor LY83583 (LY, 2.5 μMg), the inhibitor of Tyr-protein kinase at low concentrations and of protein kinase G (PKG) at higher concentrations lavendustin A (LAV, 0.1 and 0.5 μMg), the PKG inhibitor KT5823 (2.0 μMg), the protein kinase C (PKC) inhibitor staurosporin (STAU, 2.5 μMg), the inhibitor of calcium/ calmodulin protein kinase II (CaMKII) KN62 (3.6 μMg), the protein kinase A (PKA) inhibitor KT5720 (0.5 μMg), and the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059 (PD, 0.05 μMg). PD was dissolved in saline; all the other drugs were dissolved in 20% dimethyl sulfoxide. In all cases the drugs affected LTM as had been described in previous papers. The drugs affected STM and LTM differentially depending on the brain structure into which they were infused. STM was inhibited by KT5720, LY, and PD given into CA1 and by STAU and KT5720 given into the entorhinal cortex. PD given into the entorhinal cortex enhanced STM. LTM was inhibited by STAU, KN62, KT5720, KT5823, and LAV (0.5 μMg) given into CA1 and by STAU, KT5720, and PD given into the entorhinal cortex. The results suggest that STM and LTM involve different physiological mechanisms but are to an extent linked. STM appears to require PKA, guanylyl cyclase, and MAPKK activity in CA1 and PKA and PKC activity in the entorhinal cortex; MAPKK seems to play an inhibitory role in STM in the entorhinal cortex. In contrast, LTM appears to require PKA and PKC activity in both structures, guanylyl cyclase, PKG, and CaMKII activity in CA1, and MAPKK activity in the entorhinal cortex.

In vitro phosphorylation of cytoskeletal proteins in the rat cerebral cortex is decreased by propionic acid.

In the present study we demonstrate that propionic acid (PA), a metabolite that accumulates in large amounts in propionic acidemia, is able to decrease in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulin. Considering that the endogenous phosphorylating system associated with the cytoskeletal fraction contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin protein kinase II (CaMKII), and protein phosphatase 1 (PP1), we first assayed the effect of the acid on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. Results demonstrated that the acid totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, PA partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. In addition, we demonstrated that PA totally inhibited in vitro dephosphorylation of neurofilament subunits and tubulins mediated by PP1 in brain slices pretreated with the acid. Taken together, these results demonstrate that PA inhibits the in vitro activities of PKA, CaMKII, and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats. This study suggests that PA at the same concentrations found in tissues from propionic acidemic children may alter phosphorylation of cytoskeletal proteins, which may contribute to the neurological dysfunction characteristic of propionic acidemia.