Antisense suppression of GABA(A) receptor beta subunit levels in cultured cerebellar granule neurons demonstrates their importance in receptor expression

GABA(A) receptors in the CNS are pentameric molecules composed of alpha, beta, gamma, delta, epsilon and theta subunits. Studies on transfected cells have shown that GABA(A) receptor beta subunit isoforms can direct alpha1 subunit localization within the cell. To examine the role of selected subunits in governing GABA(A) receptor expression in neurons, cultures of rat cerebellar granule cells were grown with antisense or sense oligodeoxynucleotides (ODNs) specific for the alpha 1, beta 2 or gamma 2 subunits. These subunits are all expressed in granule neurons where they are thought to contribute to an abundant receptor type. Following ODN treatment, subunit expression and distribution were examined by western blotting, immunocytochemistry and RT-PCR. Treatment of the cultures with the antisense, but not the corresponding sense, ODNs reduced the levels of the targeted subunit polypeptides. In addition, the beta 2 antisense ODN reduced the level of the alpha1 subunit polypeptide without altering the level of its mRNA. In contrast, treatment with the beta 2 subunit antisense ODN did not alter gamma 2 subunit polypeptide expression, distribution or mRNA level. These findings suggest that the alpha1 subunit requires a beta subunit for assembly into GABA(A) receptors in cerebellar granule neurons.     

Modulation of cytolytic T lymphocyte functions by an inhibitor of serine/threonine phosphatase, okadaic acid. Enhancement of cytolytic T lymphocyte-mediated cytotoxicity.

A specific and potent inhibitor of protein phosphatases 1 and 2A, okadaic acid (OA), and its inactive analog, tetramethyl ether (OA-TME), were tested in the cytotoxicity and granule exocytosis assays of CTL activation. At low concentrations OA enhanced, whereas at higher concentrations OA inhibited, CTL responses. The Ag-specific and retargeted cytotoxicity, granule exocytosis induced by target cell (TC), anti-TCR mAb, or PMA and A23187, and conjugate formation with TC were inhibited by pretreatment of CTL with OA as expected if protein phosphatases and protein dephosphorylation were indeed involved in the TCR-mediated signal transduction and effector responses of CTL. Cytotoxicity and granule exocytosis were unaffected by pretreatment of CTL with OA-TME. The inhibitory effect of OA on the exocytic response of CTL induced by TC and anti-TCR mAb can be dissociated from the inhibition of the response to PMA and A23187, suggesting the involvement of a serine and/or threonine protein phosphatase in the early events of transmembrane signaling. At lower concentrations, OA, but not OA-TME, was able to enhance the Ag-specific cytotoxicity and TC-induced exocytosis from CTL clones. The enhancement of these TCR-mediated responses of CTL was observed only if the activation was induced by the Ag on the TC surface, because OA did not enhance either the anti-TCR mAb-induced exocytosis of granules from the CTL clone or lysis of the Ag-nonbearing TC by CTL in a retargeting assay. The biphasic character of the effects of OA on CTL-TC interactions suggests the existence of at least two functionally distinct phosphatases in CTL. The ability of OA to enhance the Ag-specific response is unique and indicates the presence of an inhibitory phosphoprotein phosphatase that should be considered as a participant in the down-regulation of the cell-cell interactions between CTL and TC. The inhibitory effects of OA on both TC-induced and anti-TCR mAb-triggered CTL responses at higher concentrations point to the importance of yet another phosphatase in the CTL-TC interactions and in the TCR-mediated transmembrane signaling. The use of OA may help to decipher the details of biochemical changes involved in T lymphocyte effector functions.     

Functional regulation of L-type calcium channels via protein kinase A-mediated phosphorylation of the beta(2) subunit

Activation of protein kinase A (PKA) through the beta-adrenergic receptor pathway is crucial for the positive regulation of cardiac L-type currents; however it is still unclear which phosphorylation events cause the robust regulation of channel function. In order to study whether or not the recently identified PKA phosphorylation sites on the beta(2) subunit are of functional significance, we coexpressed wild-type (WT) or mutant beta(2) subunits in tsA-201 cells together with an alpha(1C) subunit, alpha(1C)Delta1905, that lacked the C-terminal 265 amino acids, including the only identified PKA site at Ser-1928. This truncated alpha(1C) subunit was similar to the truncated alpha(1C) subunit isolated from cardiac tissue not only in size ( approximately 190 kDa), but also with respect to its failure to serve as a PKA substrate. In cells transfected with the WT beta(2) subunit, voltage-activated Ba(2+) currents were significantly increased when purified PKA was included in the patch pipette. Furthermore, mutations of Ser-478 and Ser-479 to Ala, but not Ser-459 to Ala, on the beta(2) subunit, completely abolished the PKA-induced increase of currents. The data indicate that the PKA-mediated stimulation of cardiac L-type Ca(2+) currents may be at least partially caused by phosphorylation of the beta(2) subunit at Ser-478 and Ser-479.     

Triggering of CD8+ cytotoxic T lymphocytes via CD3-epsilon differs from triggering via alpha/beta T cell receptor. CD3-epsilon-induced cytotoxicity occurs in the absence of protein kinase C and does not result in exocytosis of serine esterases.

The requirement for protein kinase C (PKC) during triggering of murine CD8+ CTL was investigated. To this, CTL were depleted for PKC by pretreatment with PMA. This procedure neither influenced alpha/beta-TCR, CD3-epsilon, CD8, CD2, and lymphocyte function-associated Ag-1 expression, nor CTL-target cell conjugate formation. Although cytolytic effector function of PKC-depleted CTL triggered via alpha/beta-TCR structures was completely inhibited, target cell lysis induced via CD3-epsilon remained unaffected. Furthermore this PKC-independent cytolysis pathway was not associated with the release of serine esterases. Analyses at the clonal level revealed that PKC depletion blocked the cytolytic response of up to 95% of alpha/beta-TCR triggered CTL clones. The data suggest the existence of a distinct signaling pathway triggered via CD3-epsilon that is not associated with exocytosis of serine esterases and probably independent of PKC.     

Regulation of cardiac L-type Ca2+ channel by coexpression of G(alpha s) in Xenopus oocytes

Activation of G(alpha s) via beta-adrenergic receptors enhances the activity of cardiac voltage-dependent Ca2+ channels of the L-type, mainly via protein kinase A (PKA)-dependent phosphorylation. Contribution of a PKA-independent effect of G(alpha s) has been proposed but remains controversial. We demonstrate that, in Xenopus oocytes, antisense knockdown of endogenous G(alpha s) reduced, whereas coexpression of G(alpha s) enhanced, currents via expressed cardiac L-type channels, independently of the presence of the auxiliary subunits alpha2/delta or beta2A. Coexpression of G(alpha s) did not increase the amount of alpha1C protein in whole oocytes or in the plasma membrane (measured immunochemically). Activation of coexpressed beta2 adrenergic receptors did not cause a detectable enhancement of channel activity; rather, a small cAMP-dependent decrease was observed. We conclude that coexpression of G(alpha s), but not its acute activation via beta-adrenergic receptors, enhances the activity of the cardiac L-type Ca2+ channel via a PKA-independent effect on the alpha1C subunit.     

Cellular concentrations of protein kinase A modulate prostaglandin and cAMP induction of alkaline phosphatase.

The relationship between the concentration of cAMP-dependent protein kinase (PKA) activity and the induction of alkaline phosphatase (AP) was examined in transfected L cell lines with altered PKA levels. C alpha 12 cells were generated by transfecting mouse L cells with an expression vector coding for the mouse C alpha catalytic subunit of PKA and were shown to contain 2.5-fold more PKA activity than L cells. RAB10 cells were generated by transfection with an expression vector for a mutant regulatory subunit and had 10-fold lower levels of PKA activity than L cells. AP induction by 8-chlorophenylthio-cAMP (CPT-cAMP) was found to be 2-fold greater in C alpha 12 cells than in L cells, while RAB10 cells lacked any induction of AP in response to CPT-cAMP. Northern blot and solution hybridization analyses of AP mRNA showed that induced AP mRNA levels were comparable in C alpha 12 and in L cells. Western blot analysis demonstrated that AP protein levels were greater in C alpha 12 cells and suggested that the increased AP protein level resulted from either increased stability of the AP protein or increased rate of translation of the AP mRNA. In contrast, Northern blot analysis of the RAB10 cells failed to detect AP mRNA after CPT-cAMP treatment and suggested that PKA is required for induction of AP mRNA. Stimulation of endogenous cAMP levels by treatment with prostaglandin E1 gave similar effects on AP activity as those seen with CPT-cAMP. These results indicate that cellular levels of PKA can determine the magnitude of cellular response to hormonal stimulation and also suggest that PKA can regulate AP gene expression at both the level of the AP mRNA and AP protein.     

Regulation on the expression and N-glycosylation of integrins by N-acetylglucosaminyltransferase V

The expressions of integrin alpha5, beta1, and alpha6 were studied in H7721 cells by means of flow cytometric and RT-PCR method after transfected with sense and antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V). The transfected cells were characterized by Northern blot. It was found that the order of expression from high to low was beta1>alpha5>alpha6. Transfection of sense GnT-V up-regulated alpha5 and alpha6, but not beta1 subunit, while antisense GnT-V down-regulated alpha5 and beta1, but not alpha6. The alterations of surface integrin subunits were quite compatible with the changes of their mRNAs. Using enzyme-labeled lectin analysis, it was shown that alpha5 subunit contained only C(2)C(2) biantennary N-glycan, which was not regulated by sense and antisense GnT-V. In contrast, beta1 subunit contained both biantennary and tri-/tetra-antennary N-glycans with GlcNAcbeta1,6Manalpha1,6-branch, and the latter was up- and down-regulated by the sense and antisense GnT-V, respectively. Therefore, the amount of biantennary N-glycans on beta1 subunit, but not the integrin protein, was correlated to the cell adhesion to fibronectin and laminin, which was reduced and elevated in the sense and antisense GnT-V-transfected cells, respectively, as we previously reported.     

Protein kinase A regulates GATA-3-dependent activation of IL-5 gene expression in Th2 cells

Treatment of Th cells with compounds that elevate cAMP levels augments Th2-type lymphokine expression, in particular the synthesis of IL-5. Using primary murine CD4(+) T lymphocytes, we show in this study that inhibition of protein kinase A (PKA) activity in Th2 effector cells impairs IL-5 synthesis, whereas the expression of PKA catalytic subunit alpha enhances IL-5 synthesis in Th0 cells. In addition, we observed by coexpression of PKA catalytic subunit and GATA-3 in Th1 cells that the stimulatory effect of PKA is dependent on GATA-3 activity. These data demonstrate that activation of PKA in Th effector cells induces the IL-5 gene expression in a GATA-3-dependent manner.     

Identification and characterization of novel PKA holoenzymes in human T lymphocytes

Cyclic AMP-dependent protein kinase (PKA) is a holoenzyme that consists of a regulatory (R) subunit dimer and two catalytic (C) subunits that are released upon stimulation by cAMP. Immunoblotting and immunoprecipitation of T-cell protein extracts, immunofluorescence of permeabilized T cells and RT/PCR of T-cell RNA using C subunit-specific primers revealed expression of two catalytically active PKA C subunits C alpha1 (40 kDa) and C beta2 (47 kDa) in these cells. Anti-RI alpha and Anti-RII alpha immunoprecipitations demonstrated that both C alpha1 and C beta2 associate with RI alpha and RII alpha to form PKAI and PKAII holoenzymes. Moreover, Anti-C beta2 immunoprecipitation revealed that C alpha1 coimmunoprecipitates with C beta2. Addition of 8-CPT-cAMP which disrupts the PKA holoenzyme, released C alpha1 but not C beta2 from the Anti-C beta2 precipitate, indicating that C beta2 and C alpha1 form part of the same holoenzyme. Our results demonstrate for the first time that various C subunits may colocate on the same PKA holoenzyme to form novel cAMP-responsive enzymes that may mediate specific effects of cAMP.     

Imidazoline NNC77-0074 stimulates Ca2+-evoked exocytosis in INS-1E cells by a phospholipase A2-dependent mechanism

We have previously demonstrated that the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) increases insulin secretion from pancreatic beta-cells by stimulation of Ca(2+)-dependent exocytosis. Using capacitance measurements, we now show that NNC77-0074 stimulates exocytosis in clonal INS-1E cells. NNC77-0074-stimulated exocytosis was antagonised by the cytoplasmic phospholipase A(2) (cPLA(2)) inhibitors ACA and AACOCF(3) and in cells treated with antisense oligonucleotide against cPLA(2)alpha. NNC77-0074-evoked insulin secretion was likewise inhibited by ACA, AACOCF(3), and cPLA(2)alpha antisense oligonucleotide treatment. In pancreatic islets NNC77-0074 stimulated PLA(2) activity. We propose that cPLA(2)alpha plays an important role in the regulation of NNC77-0074-evoked exocytosis in insulin secreting beta-cells.     

Effects of treatment with GABA(A) receptor subunit antisense oligodeoxynucleotides on GABA-stimulated 36Cl- influx in the rat cerebral cortex

GABA(A) receptor function was studied in cerebral cortical vesicles prepared from rats after intracerebroventricular microinjections of antisense oligodeoxynucleotides (aODNs) for alpha1, gamma2, beta1, beta2 subunits. GABA(A) receptor alpha1 subunit aODNs decreased alpha1 subunit mRNA by 59+/-10%. Specific [3H]GABA binding was decreased by alpha1 or beta2 subunit aODNs (to 63+/-3% and 64+/-9%, respectively) but not changed by gamma2 subunit aODNs (94+/-5%). Specific [3H]flunitrazepam binding was increased by alpha1 or beta2 subunit aODNs (122+/-8% and 126+/-11%, respectively) and decreased by gamma2 subunit aODNs (50+/-13%). The "knockdown" of specific subunits of the GABA(A )receptor significantly influenced GABA-stimulated 36Cl- influx. Injection of alpha1 subunit aODNs decreased basal 36Cl- influx and the GABA Emax; enhanced GABA modulation by diazepam; and decreased antagonism of GABA activity by bicuculline. Injection of gamma2 subunit aODNs increased the GABA Emax; reversed the modulatory efficacy of diazepam from enhancement to inhibition of GABA-stimulation; and reduced the antagonist effect of bicuculline. Injection of beta2 subunit aODNs reduced the effect of diazepam whereas treatment with beta1 subunit aODNs had no effect on the drugs studied. Conclusions from our studies are: (1) alpha1 subunits promote, beta2 subunits maintain, and gamma2 subunits suppress GABA stimulation of 36Cl- influx; (2) alpha1 subunits suppress, whereas beta2, and gamma2 subunits promote allosteric modulation by benzodiazepines; (3) diazepam can act as an agonist or inverse agonist depending on the relative composition of the receptor subunits: and (4) the mixed competitive/non-competitive effects of bicuculline result from activity at alpha1 and gamma2 subunits and the lack of activity at beta1 and beta2 subunits.     

Decreased expression of the type I isozyme of cAMP-dependent protein kinase in tumor cell lines of lung epithelial origin

A spontaneous transformant derived from a mouse lung epithelial cell line exhibited decreased cAMP-dependent protein kinase (PKA) activity. DEAE column chromatography demonstrated that this was caused by specific loss of the type I PKA isozyme (PKA I). Western immunoblot analysis indicated that indeed several mouse lung tumor-derived cell lines and spontaneous transformants of immortalized, nontumorigenic lung cell lines contained less PKA I regulatory subunit (RI) protein than normal cell lines. PKA II regulatory subunit protein differed only slightly among cell lines and showed no conspicuous trend between normal and neoplastic cells. The decrease in RI was apparently concomitant with decreased catalytic (C) subunit levels in neoplastic cells since no free catalytic subunit activity was detected by DEAE chromatography. Northern blot analysis using RI alpha and C alpha cDNA probes showed that the levels of RI alpha and C alpha mRNAs paralleled their intracellular protein concentrations; neoplastic cell lines contained significantly less RI alpha and C alpha mRNAs than the normal cell line. The decreased expression of both RI and C subunits therefore results in a net decrease of PKA I in neoplastic lung cells, an isozymic difference which may account for the differential effects of cAMP analogs on cell growth and differentiation in normal and neoplastic cells.     

The level of friend retrovirus replication determines the cytolytic pathway of CD8+ T-cell-mediated pathogen control

Cytotoxic T cells (CTL) play a central role in the control of viral infections. Their antiviral activity can be mediated by at least two cytotoxic pathways, namely, the granule exocytosis pathway, involving perforin and granzymes, and the Fas-FasL pathway. However, the viral factor(s) that influences the selection of one or the other pathway for pathogen control is elusive. Here we investigate the role of viral replication levels in the induction and activation of CTL, including their effector potential, during acute Friend murine leukemia virus (F-MuLV) infection. F-MuLV inoculation results in a low-level infection of adult C57BL/6 mice that is enhanced about 500-fold upon coinfection with the spleen focus-forming virus (SFFV). Both the low- and high-level F-MuLV infections generated CD8+ effector T cells that were essential for the control of viral replication. However, the low-level infection induced CD8+ T cells expressing solely FasL but not the cytotoxic molecules granzymes A and B, whereas the high-level infection resulted in induction of CD8+ effector T cells secreting molecules of the granule exocytosis pathway. By using knockout mouse strains deficient in one or the other cytotoxic pathway, we found that low-level viral replication was controlled by CTL that expressed FasL but control of high-level viral replication required perforin and granzymes. Additional studies, in which F-MuLV replication was enhanced experimentally in the absence of SFFV coinfection, supported the notion that only the replication level of F-MuLV was the critical factor that determined the differential expression of cytotoxic molecules by CD8+ T cells and the pathway of CTL cytotoxicity.