Pharmacological strategies for neuroprotection in traumatic brain injury.

Traumatic brain injury affects over a million Americans annually, but pharmacological therapy remains limited. Current standards of care in acute, subacute and chronic phases of injury are primarily supportive. This review discusses pharmacological strategies and future directions in patient treatment emphasizing pleiotropic agents targeting inflammation, oxidative damage, and glutamate excitotoxicity.

Structural properties of the NMDA receptor and the design of neuroprotective therapies.

NMDA receptors are linked to neuronal loss in stroke and neurodegeneration because their activation can trigger excitotoxic Ca(2+) dysregulation. Accordingly, NMDA receptor antagonists are neuroprotective, providing a rationale for their clinical application. However, side effects often outweigh benefits. Herein we highlight structural properties in receptors that are used in drug development.

Cyclin-dependent kinase inhibitors: cancer killers to neuronal guardians.

The development of small molecule kinase inhibitors as potential cancer therapeutics is an area of intense interest, and a subset of these agents target cyclin-dependent kinase (CDK) activity. Ten distinct CDKs (1-9, 11), when paired with their cyclin activators, are integral to such diverse processes as cell cycle control, neuronal development, and transcriptional regulation. Mutation and/or aberrant expression of certain CDKs and their regulatory counterparts are associated with uncontrolled proliferation and tumorigenesis. As such, CDK selective inhibitors (CDKIs) that attenuate or prevent tumor growth have been developed. Recently, interest in the therapeutic potential of CDKIs has expanded to include neurodegenerative diseases, where dysregulated CDK activity has been linked to the pathogenesis of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and stroke. Specifically, aberrant activation of cell cycle CDKs or CDK5 is associated with apoptosis and neuronal dysfunction in response to various neuronal stressors. To date, CDKIs have shown promise as neuroprotective agents in the research laboratory and, in the future, may prove useful in the neurology clinic.

2,2',3,3',4,4'-Hexahydroxy-1,1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE)-induced neuronal apoptosis independent of classical protein kinase C alpha or gamma inhibition.

Protein kinase C (PKC) isozymes constitute a family of at least 12 structurally related serine-threonine kinases that are differentially regulated and localized, and are presumed to mediate distinct intracellular functions. To explore their roles in intact cells, investigators are developing cell-permeable, isoform-selective inhibitors. 2,2',3,3',4,4'-Hexahydroxy-1, 1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE) is reported to be a selective inhibitor of PKC alpha and gamma with IC(50) values of 43 and 50 microM, respectively, using an in vitro assay. However, data examining the potency and selectivity of HBDDE in intact cells are lacking. Employing rodent cerebellar granule neurons as a model system, we investigated the effects of HBDDE using cell survival as a functional end-point. HBDDE induced an apoptotic form of cell death that was dependent upon protein synthesis and included activation of a terminal executioner of apoptosis, caspase 3. The concentration of HBDDE required for half-maximal cell death was less than 10 microM ( approximately 5-fold less than the reported IC(50) values for PKC alpha and gamma in vitro). Furthermore, HBDDE induced apoptosis even after phorbol-ester-mediated down-regulation of PKC alpha and gamma, indicating that this effect is independent of these isoforms. Consistent with this, 2-[1-(3-dimethylaminopropyl) indol-3-yl]-3-(indol-3-yl)-maleimide (GF 109203X), a general inhibitor of all classical and some novel PKCs, did not interfere with survival. Thus, HBDDE should not be used as an isoform-selective inhibitor of PKC alpha or gamma in intact cells. Nevertheless, identification of its target in granule neurons will provide valuable information about survival pathways.

Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations.

Multiple isoforms of type II Ca(2+)-calmodulin-dependent kinase (CaM KII) are composed of two major neuron-specific subunits, designated alpha and beta, and two less well-characterized subunits that are also expressed in non-neuronal tissues, designated delta and gamma. Regulated expression of these 4 gene products, and several variants produced by alternative splicing, shows temporal and regional specificity and influences intracellular targeting. We used immunoblotting and RT-PCR to analyze subunit and variant expression and distribution in cultured cerebellar astrocytes and neurons, and whole cerebellar cortex from rodent brain. The data indicate that: (i) astrocytes express a single splice variant of delta, namely delta(2); (ii) like neurons, astrocytes express two forms of CaM KII gamma; gamma(B) and gamma(A); (iii) these CaM KII variants are enriched in the supernate fraction in astrocytes, and the particulate fraction in neurons; (iv) unlike neurons, astrocytes do not express detectable levels of alpha or beta subunits or their respective splice variants. The results indicate that neurons and astrocytes express distinct CaM KII subunits and variants that localize to distinct subcellular compartments and, by inference, exert distinct cellular functions.

Ca2+ and pH modulate alternative splicing of exon 5 in NMDA receptor subunit 1.

RT-PCR and intracellular Ca2+ measurements were used to identify factors that modulate alternative splicing of exon 5 in the NMDA receptor transcript encoding NR1, in cultured cerebellar granule neurons. Although cells grown in media containing 5 mM KCl demonstrate compromised survival, they show the predicted developmental transition from NR1a (-exon 5) to NR1b (+exon 5) mRNA expression. This transition was blocked under culture conditions that promote survival; inclusion or exclusion of exon 5 is a reversible process that is sensitive to alterations in Ca2+ and pH. We conclude that alternative splicing of NR1 pre-mRNA transcripts may be regulated by developmental cues that modulate the degree of glutamate receptor activation.

Developmental aspects of NMDA receptor function.

Glutamate is the major excitatory neurotransmitter in mammalian synapses. It binds to three classes of predominantly postsynaptic ionotropic receptors to activate receptor-associated channels, and a class of metabotropic receptors to activate G-protein mediated transduction pathways. The N-methyl-D-aspartate (NMDA) receptor (NR) is distinctive in being both ligand and voltage-gated, and selectively permeable to Ca2+. As a consequence, NR-mediated alterations in intracellular Ca2+ levels regulate a variety of signaling pathways, ranging from localized, acute effects on receptor and channel activities to long-term effects on nuclear gene transcription. Regulated expression, assembly, and modulation of distinct heteromeric NR complexes comprised of different subunit combinations contributes to this functional diversity. NRs have been implicated in several developmental processes, and evidence supporting their role in migration, survival, and synaptic maturation is discussed.

Glia modulate NMDA-mediated signaling in primary cultures of cerebellar granule cells.

Excessive activation of N-methyl-D-aspartate (NMDA) receptor channels (NRs) is a major cause of neuronal death associated with stroke and ischemia. Cerebellar granule neurons in vivo, but not in culture, are relatively resistant to toxicity, possibly owing to protective effects of glia. To evaluate whether NR-mediated signaling is modulated when developing neurons are cocultured with glia, the neurotoxic responses of rat cerebellar granule cells to applied NMDA or glutamate were compared in astrocyte-rich and astrocyte-poor cultures. In astrocyte-poor cultures, significant neurotoxicity was observed in response to NMDA or glutamate and was inhibited by an NR antagonist. Astrocyte-rich neuronal cultures demonstrated three significant differences, compared with astrocyte-poor cultures: (a) Neuronal viability was increased; (b) glutamate-mediated neurotoxicity was decreased, consistent with the presence of a sodium-coupled glutamate transport system in astrocytes; and (c) NMDA- but not kainate-mediated neurotoxicity was decreased, in a manner that depended on the relative abundance of glia in the culture. Because glia do not express NRs or an NMDA transport system, the mechanism of protection is distinct from that observed in response to glutamate. No differences in NR subunit composition (evaluated using RT-PCR assays for NR1 and NR2 subunit mRNAs), NR sensitivity (evaluated by measuring NR-mediated changes in intracellular Ca2+ levels), or glycine availability as a coagonist (evaluated in the presence and absence of exogenous glycine) were observed between astrocyte-rich and astrocyte-poor cultures, suggesting that glia do not directly modulate NR composition or function. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked NMDA-mediated toxicity in astrocyte-poor cultures, raising the possibility that glia effectively reduce the accumulation of highly diffusible and toxic arachidonic acid metabolites in neurons. Alternatively, glia may alter neuronal development/phenotype in a manner that selectively reduces susceptibility to NR-mediated toxicity.

Expression and regulation of types I and II inositol 1,4,5-trisphosphate receptors in rat cerebellar granule cell preparations.

Previous studies have shown that as rat cerebellar granule cell cultures differentiate in the presence of 25 mM KCl, they "up-regulate" their ability to form inositol phosphates and release Ca2+ from internal stores in response to the activation of phosphoinositidase C-linked muscarinic and metabotropic receptors. Here we show that they simultaneously up-regulate their ability to respond to inositol 1,4,5-trisphosphate (InsP3) by increasing InsP3 receptor (InsP3R) expression. In contrast, if granule cells are maintained at the more physiological KCl concentration of 5 mM, most cells undergo apoptosis, although a significant number survive. The surviving cells, however, express few InsP3Rs, suggesting that an influx of Ca2+ through voltage-dependent channels is required for InsP3R up-regulation. In addition, we have determined that these cultures express two genetically distinct InsP3R types, but that only one, the type I receptor, is expressed in granule cells. Type II receptors are also present but are found exclusively in astrocytes, which are a minor contaminant of granule cell cultures. This segregation of InsP3R types explains a previous observation, showing that the muscarinic agonist carbachol causes the reduction or "down-regulation" of type I but not type II InsP3Rs.

Neuronal activity differentially regulates NMDA receptor subunit expression in cerebellar granule cells.

Reverse-transcription PCR assays were used to measure levels of NMDA receptor (NR) subunit mRNAs encoding splice variants of NR1 (NR1a, -exon 5; NR1b, +exon 5) and the major NR2 subunits (NR2A, NR2B, and NR2C) in dissociated cerebellar granule cell cultures. Cultures chronically exposed to 25 mM KCl or 100 microM NMDA/15 mM KCl, which promote survival by stimulating Ca2+ influx through voltage-sensitive Ca2+ channels or NRs, were compared with 5 mM KCl culture conditions, which results in limited cell survival attributable to a lower level of NR stimulation by ambient glutamate. In situ granule-cell maturation is associated with downregulation of NR2B and increases both of NR2A and NR2C and in the ratio of NR1b/NR1a mRNAs. In culture, 25 mM KCl or NMDA rapidly induced NR2A and downregulated NR2B, followed by gradual induction of NR2C. In 5 mM KCl, a similar, rapid increase in NR2A was observed, but disappearance of NR2B occurred over a longer time course. By 9-12 d in vitro in 5 mM KCl, the relative proportions of all three NR2 mRNAs in surviving cells were not significantly different from cells cultured in 25 mM KCl. NR1a mRNA predominated at every stage of culture in 25 mM KCl or NMDA, however, whereas gradual induction of the mature-form NR1b was observed in 5 mM KCl. Although using high potassium- or NMDA-containing media enhanced granule cell survival, it did not reproduce the pattern of expression of NR mRNAs observed in situ, whereas this pattern was observed in granule cells surviving in 5 mM KCl.

Arachidonic acid, but not sodium nitroprusside, stimulates presynaptic protein kinase C and phosphorylation of GAP-43 in rat hippocampal slices and synaptosomes.

Activation of protein kinase C (PKC) and phosphorylation of its presynaptic substrate, the 43-kDa growth-associated protein GAP-43, may contribute to the maintenance of hippocampal long-term potentiation (LTP) by enhancing the probability of neurotransmitter release and/or modifying synaptic morphology. Induction of LTP in rat hippocampal slices by high-frequency stimulation of Schaffer collateral-CA1 synapses significantly increased the PKC-dependent phosphorylation of GAP-43, as assessed by quantitative immunoblotting with a monoclonal antibody that recognizes an epitope that is specifically phosphorylated by PKC. The stimulatory effect of high-frequency stimulation on levels of immunoreactive phosphorylated GAP-43 was not observed when 4-amino-5-phosphonovalerate (50 microM), an N-methyl-D-aspartate (NMDA) receptor antagonist, was bath-applied during the high-frequency stimulus. This observation supports the hypothesis that a retrograde messenger is produced postsynaptically following NMDA receptor activation and diffuses to the presynaptic terminal to activate PKC. Two retrograde messenger candidates--arachidonic acid and nitric oxide (sodium nitroprusside was used to generate nitric oxide)--were examined for their effects in hippocampal slices on PKC redistribution from cytosol to membrane as an indirect measure of enzyme activation and PKC-specific GAP-43 phosphorylation. Bath application of arachidonic acid, but not sodium nitroprusside, at concentrations that produce synaptic potentiation (100 microM and 1 mM, respectively) significantly increased translocation of PKC immunoreactivity from cytosol to membrane as well as levels of immunoreactive, phosphorylated GAP-43. The stimulatory effect of arachidonic acid on GAP-43 phosphorylation was also observed in hippocampal synaptosomes. These results indicate that arachidonic acid may contribute to LTP maintenance by activation of presynaptic PKC and phosphorylation of GAP-43 substrate. The data also suggest that nitric oxide does not activate this signal transduction system and, by inference, activates a distinct biochemical pathway.

Chronic mild acidosis specifically reduces functional expression of N-methyl-D-aspartate receptors and increases long-term survival in primary cultures of cerebellar granule cells.

Previous studies suggest that chronic depolarization by addition of 25 mM KCl or N-methyl-D-aspartate to primary cultures of cerebellar granule cells promotes expression of the N-methyl-D-aspartate subtype of glutamate receptor, as determined by electrophysiological responsiveness and susceptibility to excitotoxicity. Recent studies have demonstrated that acute mild acidosis reduces N-methyl-D-aspartate receptor channel activity by a non-competitive action of H+ on an extracellular site of the receptor channel complex. Since the level of N-methyl-D-aspartate receptor expression in granule cell cultures is activity-dependent, we examined whether chronic mildly acidotic culture conditions would selectively diminish the level of N-methyl-D-aspartate responsiveness in granule cells, in effect producing a functional level of expression more comparable to that observed in vivo. To test this, cerebellar granule cells from eight-day neonatal rats were grown in an HCO3-buffered medium containing elevated K+ (25 mM KCl) either under standard conditions (95% air/5% CO2, pH 7.4), or under chronic mildly acidotic conditions (90% air/10% CO2, estimated pH of 7.1). Glutamate receptor subtype expression was subsequently assessed using standard neurotoxicity assays, a quantitative immunoblotting assay for N-methyl-D-aspartate receptors and whole cell patch clamp recordings. Cells grown in the 10% CO2 environment exhibited a significant reduction in susceptibility to L-glutamate neurotoxicity (at least 10-fold), but not kainate-induced neurotoxicity, relative to cells grown in 5% CO2. In both culture conditions, L-glutamate- and kainate-induced toxicity were mediated by activation of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors, respectively, as determined by the sensitivity of agonist-induced toxicity to specific receptor antagonists. Using polyclonal antibodies generated against a peptide sequence recognizing five of eight splice variants in the common "R1" subunit of N-methyl-D-aspartate receptors, a 31% reduction in the amount of immunoreactive protein was observed in membrane preparations from cells grown in 10% CO2, relative to the amount detected in cells grown in 5% CO2. Moreover, perfusion of cells with glutamate (50 microM) in a nominally Mg(2+)-free solution containing glycine (2 microM) elicited N-methyl-D-aspartate antagonist-sensitive inward currents in proportionately fewer cells cultured in 10% CO2, relative to cells cultured in 5% CO2. Long-term survival was also significantly enhanced in cells exposed chronically to mild acidotic culture conditions, relative to cells grown under standard pH conditions (22 days, 10% CO2 vs 16 days, 5% CO2).(ABSTRACT TRUNCATED AT 400 WORDS)

Distinct mode of microtubule-associated protein 2 expression in the neuroblastoma/glioma cell line 108CC15/NG108-15.

The properties of microtubule-associated protein-2 (MAP-2) expression were examined in a transformed cell line, and compared to neurons from rodent brain where evidence supports both transcriptional and nontranscriptional regulation of MAP-2 synthesis. A monoclonal antibody that recognizes a common epitope in the adult (HMW MAP-2) and juvenile (MAP-2c) forms was used in an immunoblotting assay to assess the protein levels in actively dividing and differentiated neuroblastoma/glioma (108CC15, also designated NG108-15) cells. Multiply-phosphorylated MAP-2c was the predominant form in actively dividing cells, whereas HMW MAP-2 predominated in differentiated cells, which exhibited several other neuronal-like properties. A progressive increase in the levels of immunoreactive HMW MAP-2 was observed with increasing days of cell differentiation using dBcAMP as the inducing agent. However, the absolute levels of both HMW MAP-2 and MAP-2c in NG108-15 cells were significantly lower (at least 10-fold) than levels measured in rodent brain. To assess whether there are correspondingly lower levels of HMW MAP-2 and MAP-2c mRNAs in NG108-15 cells, relative to rodent brain, a highly sensitive RNA amplification assay (reverse transcription-polymerase chain reaction; RT-PCR) was developed. Oligonucleotide primers were designed to specify either HMW MAP-2 mRNA or MAP-2c mRNA, and whole tissue RNA extracted from adult and neonatal rodent brain was used to verify the reliability of the RT-PCR assay. Accordingly, PCR products of the predicted size, specificity, and abundance were obtained, with similar levels of HMW MAP-2 mRNA and proportionately higher levels of MAP-2c mRNA in neonatal brain, relative to adult brain. MAP-2c mRNA was the predominant transcript in actively dividing NG108-15 cells, and the amount of HMW MAP-2 mRNA gradually increased and became the predominant transcript in cells exposed to dBcAMP for 6-9 days. Thus, the observed changes in MAP-2-specific mRNAs during differentiation paralleled changes in expressed protein, suggesting that MAP-2 synthesis in NG108-15 cells is transcriptionally controlled. However, the levels of both MAP-2 mRNAs in NG108-15 cells were comparable to levels in rodent brain, despite the fact that MAP-2 protein levels are at least 10-fold lower in NG108-15 cells. These data suggest that the low levels of HMW MAP-2 and MAP-2c protein expression in NG108-15 cells are not due to correspondingly lower levels of MAP-2 mRNAs, and that transformed neuronal cell lines demonstrate a unique mode of MAP-2 regulation.

A structural basis for the unequal sensitivity of the major cardiac and liver gap junctions to intracellular acidification: the carboxyl tail length.

The regulation of junctional conductance (Gi) of the major cardiac (connexin43; Cx43) and liver (connexin32; Cx32) gap junction proteins by intracellular hydrogen ion concentration (pH; pHi), as well as well as that of a truncation mutant of Cx43 (M257) with 125 amino acids deleted from the COOH terminus, was characterized in pairs of Xenopus laevis oocytes expressing homologous channels. Oocytes were injected with 40 nl mRNAs (2 micrograms/microliters) encoding the respective proteins; subsequently, cells were stripped, paired, and incubated for 20-24 h. Gj was measured in oocyte pairs using the dual electrode voltage-clamp technique, while pHi was recorded simultaneously in the unstimulated cell by means of a proton-selective microelectrode. Because initial experiments showed that the pH-sensitive microelectrode responded more appropriately to acetate than to CO2 acidification, oocytes expressing Cx32 and wild type and mutant Cx43 were exposed to a sodium acetate saline, which was balanced to various levels of pH using NaOH and HCl. pH was changed in a stepwise manner, and quasi-steady-state Gj -pHi relationships were constructed from data collected at each step after both Gj and pHi had reached their respective asymptotic values. A moderate but significant increase of Gj was observed in Cx43 pairs as pHi decreased from 7.2 to 6.8. In both Cx32 and M257 pairs, Gj increased significantly over a wider pH range (i.e., between 7.2 and 6.3). Further acidification reversibly reduced Gj to zero in all oocyte pairs. Pooled data for the individual connexins obtained during uncoupling were fitted by the Hill equation; apparent 50%-maximum (pK;pKa) values were 6.6 and 6.1 for Cx43 and Cx32, respectively, and Hill coefficients were 4.2 for Cx43 and 6.2 for Cx32. Like Cx32, M257 had a more acidic pKa (6.1) and steeper Hill coefficient (6.0) than wild type Cx43. The pKa and Hill coefficient of M257 were very similar to those of Cx32. These experiments provide the first direct comparison of the effects of acidification on Gj in oocyte pairs expressing Cx43 or Cx32. The results indicate that structural differences in the connexins are the basis for their unequal sensitivity to intracellular acidification in vivo. The data further suggest that a common pH gating mechanism may exist between amino acid residues 1 and 256 in both Cx32 and Cx43. However, the longer carboxyl tail of Cx43 relative to Cx32 or M257 provides additional means to facilitate acidification-induced gating; its presence shifts the pKa from 6.1 (Cx32 and M257) to 6.6 (Cx43) in the conductance of these channels.

Demonstration of presynaptic protein kinase C activation following long-term potentiation in rat hippocampal slices.

Pharmacological and biochemical evidence implicate the Ca2+ and phospholipid-dependent protein kinase C in long-term potentiation. The in vitro hippocampal slice preparation was used to demonstrate redistribution of protein kinase C from cytosol to membrane and protein kinase C-dependent phosphorylation of the presynaptic growth-associated protein-43 substrate following long-term potentiation induction in area CA1. Protein kinase C translocation was assessed using both quantitative immunoblotting with a monoclonal antibody recognizing a common epitope in the alpha and beta isoforms of protein kinase C and Ca2+ and phospholipid-dependent phosphorylation of exogenous histone substrate. Slices examined 5 min after tetanus-induced spike potentiation showed no change in protein kinase C redistribution, whereas slices examined at 15-, 30- and 60-min intervals all showed a similar degree of protein kinase C translocation to membrane, although only at 15 min was the effect statistically significant. Additionally, an increase in protein kinase C-dependent growth-associated protein 43 phosphorylation was observed 10 min after high-frequency stimulation. The translocation of protein kinase C and phosphorylation of growth-associated protein 43 were dependent upon high-frequency (repetitive 400 Hz) afferent stimulation, as no effects were observed in slices receiving low-frequency (1 Hz) or no stimulation. The N-methyl-D-aspartate receptor antagonist, DL-2-amino-5-phosphonovaleric acid (50 microM), inhibited induction of long-term potentiation, redistribution of protein kinase C and phosphorylation of growth-associated protein 43. A significant redistribution of the predominantly presynaptic protein kinase C isoform, protein kinase C-alpha, was also detected 15 min after induction of long-term potentiation using an alpha-isoform-specific monoclonal antibody. These observations support a presynaptic role for protein kinase C and growth-associated protein 43 in the early maintenance phase of LTP, and further suggest that a retrograde messenger produced postsynaptically following N-methyl-D-aspartate receptor activation mediates these effects.

Immunolocalization and expression of functional and nonfunctional cell-to-cell channels from wild-type and mutant rat heart connexin43 cDNA.

The carboxyl terminal cytoplasmic domain of distinct gap junction proteins may play an important role in assembly of functional channels as well as differential responsiveness to pH, voltage, and intracellular second messengers. Oligonucleotide-directed site-specific mutagenesis in a paired Xenopus laevis oocyte expression system was used to examine the expression of mRNAs encoding wild-type and carboxyl terminal mutant connexin43 (Cx43) proteins. Oocytes were stripped, injected with mRNA or distilled water (dH2O), preincubated for 16-20 hours, and then paired for 5-10 hours; this process was followed by electrophysiological recording using the dual voltage-clamp technique. Initial experiments compared the relative junctional conductances (Gjs) in oocyte pairs expressing Cx43 (382 amino acid residues) and two truncated mutants lacking most or a portion of the cytoplasmic carboxyl terminal. The shortest mutant (M241) contained 240 amino acid residues and was devoid of all phosphorylatable serine residues in the cytoplasmic tail; its length approximated the length of liver connexin26. The longest mutant (M257) tested contained 256 amino acid residues, including two serine residues. Oocyte pairs expressing M241 yielded a Gj similar to that of oocytes injected with dH2O, whereas M257 yielded a Gj similar to that of oocytes injected with Cx43. Immunoprecipitation studies showed that Cx43, M257, and M241 proteins were readily detectable in oocytes injected with their respective mRNAs, indicating that the lack of Gj observed with the M241 mRNA was not due to reduced translation. Immunocytochemical studies revealed that wild-type and both truncated mutants were localized to the area of cell-to-cell contact between the paired oocytes, indicating that protein targeting to the membrane was not inhibited in oocytes injected with M241 mRNA. Oocyte pairs expressing mutants in which serine residues were replaced with nonphosphorylatable amino acids (serine codon No. 255 AGC was converted to GCC, alanine, designated as M255S----A, and serine codon No. 244 AGC was converted to GGC, glycine, designated as M244S----G) showed Gjs similar to M257, indicating that these serine residues and, by inference, their phosphorylation state are not critical for expression of functional channels. The importance of the length of the carboxyl terminus was assessed by comparing the Gjs in a series of mutants that were intermediate in length between M257 and M241. Gradual shortening of the carboxyl terminus produced a gradual reduction of Gj relative to M257. However, simple deletion of amino acid residues 241-257 from the wild-type Cx43 did not affect Gj relative to M257.(ABSTRACT TRUNCATED AT 400 WORDS)

Detection of mRNAs encoding distinct isoenzymes of type II calcium/calmodulin-dependent protein kinase using the polymerase chain reaction.

A modification of the polymerase chain reaction (PCR) was used to amplify nucleotide sequences encoding the 50-kDa (alpha) or 58- to 60-kDa (beta',beta) subunits of a brain-specific type II calcium/calmodulin-dependent protein kinase (CaM kinase II). Rat brain RNA from different regions and at different postnatal ages was purified, and reverse transcriptase was used to produce cDNA templates. Oligonucleotide primer pairs flanking a unique sequence in the coding region of the beta',beta subunit-specific cDNA or a unique sequence in the 3' noncoding region of the alpha subunit-specific cDNA were used to amplify sequences encoding portions of these subunits by PCR. Adult rat forebrain contained approximately three times as much alpha subunit mRNA as beta',beta subunit mRNA, whereas adult rat cerebellum contained a molar ratio of 1 alpha: 5 beta',beta. Intermediate levels of alpha and beta',beta subunit mRNAs were observed in adult pons/medulla, and in 4- and 8-day neonatal forebrain. This amplification assay was also used to demonstrate the presence of alpha subunit mRNA in cerebellar granule cells and 4-day neonatal forebrain, which was reported to be undetectable by other methods. Cerebellar granule cells contained less alpha subunit RNA relative to whole cerebellum, suggesting that this cell type expresses an isoform of CaM kinase II containing less alpha subunit protein in the holoenzyme. The observed levels of subunit-specific mRNAs were shown to parallel the levels of expressed protein subunits, suggesting that expression of kinase isoforms is transcriptionally regulated. The data also indicate that the conditions used for amplification of CaM kinase II mRNAs are semiquantitative.

Differential effects of isoquinolinesulfonamide protein kinase inhibitors on CA1 responses in hippocampal slices.

The effects of the isoquinolinesulfonamide protein kinase inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) on CA1 responses in hippocampal slices of the rat were examined to clarify their mode of action, and also to further define the role of Ca(2+) -dependent kinases in long-term potentiation. Initially, the inhibitory potencies of H-7 and HA1004 against both protein kinase C and type II Ca2+/calmodulin-dependent kinase were examined in standard in vitro phosphorylation assays. The apparent Ki values of H-7 and HA1004 for protein kinase C were 9 and 57 microM, respectively. In contrast, the Ki values of H-7 and HA1004 for type II calcium/calmodulin-dependent protein kinase were 156 and 13 microM, respectively. These results indicate that H-7 is a more effective inhibitor of protein kinase C, whereas HA1004 is a more effective inhibitor of type II calcium/calmodulin-dependent protein kinase. Following the induction of long-term potentiation, addition of 50 microM H-7 or HA1004 substantially increased the amplitude of the population spike in a control pathway, while producing no change or a slight increase in the spike amplitude in a previously potentiated long-term potentiation pathway. Moreover, H-7 (50 microM), but not HA1004, produced multiple population spikes in both pathways. Addition of a higher concentration of H-7 (300 microM) reduced the amplitude of the initial population spike but still produced multiple spikes. HA1004 (300 microM) typically produced effects similar to those observed with 50 microM H-7, increasing the amplitude of the control population spike and producing multiple spike activity in both pathways. In contrast to the differential concentration-dependent effects of H-7 on the population spike responses, qualitatively similar effects were observed at both low (50 microM) and high (300 microM) concentrations with regard to synaptic field responses. The initial slope of the population excitatory postsynaptic potential was significantly reduced by H-7, to a similar degree in both pathways. HA1004 produced a modest, but insignificant reduction in both pathways. These results, in conjunction with other reports, suggest that H-7 and HA1004 exert complex concentration-dependent effects with synchronously affect both excitatory and inhibitory synaptic transmission. We hypothesize that reduction of the population excitatory postsynaptic potential and spike (300 microM H-7) is due to reduction of excitatory inputs, whereas enhancement of the population spike amplitude (50 microM H-7) and the production of multiple spikes are due to the reduction of GABA-mediated inhibitory inputs.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental regulation of type II calcium/calmodulin-dependent kinase isoforms in rat cerebellum.

Two distinct isoforms of a Type II calcium/calmodulin-dependent protein kinase were separated from high-speed supernates (cytosol) of rat neonatal [postnatal day 10 (P10)] and adult [postnatal day 40 (P40)] cerebellum using cation-exchange chromatography. The isoenzymes contained variable amounts of three subunits of apparent Mr's of 50 kDa (alpha), 58 kDa (beta'), and 60 kDa (beta). The specific activity of calmodulin-dependent kinase (CaM kinase II) in crude homogenates increased sixfold between P10 and P40 using exogenous MAP 2 as substrate. Cytosol from cerebellum at P40 contained a predominant isoform (approximately 40% of total cytosolic activity) with a 1:5 molar ratio of alpha:beta',beta subunits that eluted with 150 mM NaCl (designated 150) and a less abundant isoform (approximately 20% of total cytosolic activity) containing a 1:8 molar ratio of alpha:beta',beta subunits that eluted with 350 mM NaCl (designated 350). In neonatal cerebellum at P10, the relative abundance of the two isoforms was reversed such that approximately 50% of the cytosolic calmodulin-dependent kinase activity was recovered in the 350 isoform, whereas only 20% of the total cytosolic kinase activity was recovered in the 150 isoform. Previous studies indicate that cerebellar granule cells may contain an all beta',beta isoform of CaM kinase II that lacks alpha subunit. Thus, to assess the cell-specific localization of kinase isoforms within cerebellum, cytosol prepared from primary cultures of rat cerebellar granule cells was applied to cation-exchange chromatography and analyzed for calmodulin-dependent kinase activity. The cells contained both isoforms of the kinase that were present in fresh tissue suggesting that granule cell-enriched cultures express all three kinase subunits. The data demonstrate that rat cerebellum contains unique mixtures of CaM kinase II isoenzymes and that their expression is developmentally regulated.