Differential Proteolysis of the Full-Length Form of the L-Type Calcium Channel α1 Subunit by Calpain

Abstract: This study examines the proteolysis of the carboxy terminal domain of the full-length (α1₂₁₂) and truncated (α1₁₉₀) forms of the rabbit skeletal muscle L-type calcium channel α1 subunit by calpain I and calpain II. Although both forms of the α1 subunit show little sensitivity to proteolysis by calpain II, α1₂₁₂ is relatively more sensitive than α1₁₉₀ to digestion by calpain I, the form of the enzyme regulated by micromolar concentrations of calcium. Calpain I cleaves a 37-kDa fragment from the C-terminus of α1₂₁₂ in a time- and concentration-dependent manner and proteolysis is independent of the α1₂₁₂ phosphorylation state. This proteolytic cleavage removes the major site of cyclic AMP-dependent phosphorylation from α1₂₁₂ and may provide a mechanism for modifying the cyclic AMP-dependent regulation of L-type calcium channels in skeletal muscle.     

Duration of synchronous cleavage cycles and rate of development at different temperatures in the Baltic herring

The duration of one synchronous cleavage cycle (τ₀) in Clupea harengus membras at different temperatures ( T ) was given by: (logτ₀)= 2.4349–0–0684T for T= 0.9–13°C, and (logτ₀)= 1.61010–oooit for t= 13–18–7°C.     

Detection of staphylococcus aureus infection by enzyme-linked immunosorbent assay and immunoblotting, using high molecular weight staphylococcal proteins

Abstract Two high molecular weight staphylococcal proteins, fibronectin-binding protein and a M _τ 200 000 protein, were investigated as antigens for serodiagnosis of staphylococcal infections. Sera from patients with staphylococcal infections and from controls were subjected to immunoblot analysis with staphylococcal lysate proteins to identify staphylococcal antigens to which patients with staphylococcal infections specifically exhibited antibodies. On such protein was found in the M _τ 200 000 region. This protein was purified and used as antigen in ElISA and compared with other antigens, namely fibronectin-binding protein(s) (FNBP, M _τ , 185 000), α-toxin and teichoic acid. Sera from patients with staphylococcal infections contained antibodies to the high molecular weight proteins in higher titers than sera from patients with non-staphylococcal infections or healthy subjects. Based on their amino-acid compositions and different abilities to bind fibronectin it was concluded that the M _τ 200 000 protein and FNBP were not identical.     

Structure and Functional Characterization of a Novel Human Low-Voltage Activated Calcium Channel

Abstract : We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca²⁺ channel α₁ subunit, α_1H, from a human medullary thyroid carcinoma cell line. The α_1H subunit is structurally similar to previously described α₁ subunits. Northern blot analysis indicates that α_1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba²⁺ currents recorded from human embryonic kidney 293 cells transiently expressing α_1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (τ = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing α_1H. Singlechannel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of ~9 pS. These channels are blocked by Ni²⁺ (IC₅₀ = 6.6 μ M ) and the T-type channel antagonists mibefradil (~50% block at 1 μ M ) and amiloride (IC₅₀ = 167 μ M ). Thus, α_1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca²⁺ channels.     

A role for p53 in the regulation of lysosomal permeability by delta 9-tetrahydrocannabinol in rat cortical neurones: implications for neurodegeneration

The psychoactive ingredient of marijuana, Δ⁹-tetrahydrocannabinol (Δ⁹-THC), can evoke apoptosis in cultured cortical neurones. Whilst the intracellular mechanisms responsible for this apoptotic pathway remain to be fully elucidated, we have recently identified a role for the CB₁ type of cannabinoid (CB) receptor and the tumour suppressor protein, p53. In the current study, we demonstrate the Δ⁹-THC promotes a significant increase in lysosomal permeability in a dose- and time-dependent manner. The increase in lysosomal permeability was blocked by the CB₁ receptor antagonist, AM251. Δ⁹-THC increased the localization of phospho-p53Ser15 at the lysosome and stimulated the release of the lysosomal cathepsin enzyme, cathepsin-D, into the cytosol. The p53 inhibitor, pifithrin-α and small interfering RNA-mediated knockdown of p53 prevented the Δ⁹-THC-mediated increase in lysosomal permeability. Furthermore, the Δ⁹-THC -mediated induction of apoptosis was abrogated by a cell-permeable cathepsin-D inhibitor (10 μM). Thus, the study demonstrates that Δ⁹-THC impacts on the lysosomal system, via p53, to evoke lysosomal instability as an early event in the apoptotic cascade. This provides evidence for a novel link between the CB₁ receptor and the lysosomal branch of the apoptotic pathway which is crucial in regulating neuronal viability following exposure to Δ⁹-THC.     

Evidence for Protein Kinase C Involvement in the Short-Term Activation by Prolactin of Tyrosine Hydroxylase in Tuberoinfundibular Dopaminergic Neurons

Abstract: The mechanism of the short-term activation by prolactin (PRL) of tyrosine hydroxylase (TH) in tuberoinfundibular dopaminergic neurons was examined in vitro on hypothalamic slices from ovariectomized rats. TH activity (determined by 3,4-dihydroxyphenylalanine accumulation in the median eminence after blockade of decarboxylase with NSD 1055) showed a dose-dependent increase within 2 h of incubation of the hypothalamic slices with PRL. To determine whether a phosphorylation process was involved in this increase in TH activity, we studied the sensitivity of the enzyme to dopamine (DA) feedback inhibition. In control median eminences, two kinetically different forms of TH coexisted, one exhibiting a K _1(DA) value of 29.92 ± 0.49 μ M , the other being × 15-fold more sensitive to DA inhibition with a K _1(DA) of 1.96 ± 0.09 μ M , likely corresponding to a phosphorylated and active form and to a nonphosphorylated and less active form, respectively. After PRL treatment, the TH form of low K _1(DA) remained unaffected, whereas the K _1(DA) of the purported active form of TH increased to 62.6 ± 0.8 μ M , suggesting an increase in the enzyme phosphorylation. This increase in the K _I(DA) of TH was selectively prevented by GF 109203X, a potent and selective inhibitor of protein kinase C, but not by a specific inhibitor of protein kinase A or calmodulin. Finally, this action of PRL could be mimicked by 12- O -tetradecan-oylphorbol 13-acetate (a direct activator of protein kinase C). These results suggest that PRL, at the median eminence level, activates TH by increasing the enzyme phosphorylation and that this action may involve an activation of protein kinase C.     

Generation and Regulation of β-Amyloid Peptide Variants by Neurons

Abstract: Studies of processing of the Alzheimer β-amyloid precursor protein (βAPP) have been performed to date mostly in continuous cell lines and indicate the existence of two principal metabolic pathways: the "β-secretase" pathway, which generates β-amyloid (Aβ_1–40/42; ∼4 kDa), and the "α-secretase" pathway, which generates a smaller fragment, the "p3" peptide (Aβ_17–40/42; ∼3 kDa). To determine whether similar processing events underlie βAPP metabolism in neurons, media were examined following conditioning by primary neuronal cultures derived from embryonic day 17 rats. Immunoprecipitates of conditioned media derived from [³⁵S]methionine pulse-labeled primary neuronal cultures contained 4- and 3-kDa Aβ-related species. Radiosequencing analysis revealed that the 4-kDa band corresponded to conventional Aβ beginning at position Aβ(Asp¹), whereas both radio-sequencing and immunoprecipitation-mass spectrometry analyses indicated that the 3-kDa species in these conditioned media began with Aβ(Glu¹¹) at the N terminus, rather than Aβ(Leu¹⁷) as does the conventional p3 peptide. Either activation of protein kinase C or inhibition of protein phosphatase 1/2A increased soluble βAPP_α release and decreased generation of both the 4-kDa Aβ and the 3-kDa N-truncated Aβ. Unlike results obtained with continuously cultured cells, protein phosphatase 1/2A inhibitors were more potent at reducing Aβ secretion by neurons than were protein kinase C activators. These data indicate that rodent neurons generate abundant Aβ variant peptides and emphasize the role of protein phosphatases in modulating neuronal Aβ generation.     

Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA_A receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA_A receptors containing the α₁ subunit, it has a lower affinity to GABA_A receptors containing α₂, α₃, or α₅ subunits and has a very weak efficacy at receptors containing the α₅ subunit. Here, we show that replacing histidine in position 105 in the α₅ subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α₅ subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α₅H105 in α₁, α₂, and α₃ are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α₁H101, α₂H101, and α₃H126 by arginine, as naturally present in α₄ and α₆, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α₅ containing receptors also for the action of zolpidem.     

ON THE SEQUENTIAL CLEAVAGE OF MYELIN BASIC PROTEIN BY CATHEPSINS A AND D

Abstract— A lysosomal carboxypeptidase (cathepsin A) known to increase in demyelination (experimental allergic encephalomyelitis and multiple sclerosis) and purified 277 fold from bovine brain failed to attack myelin basic protein. Prior treatment of basic protein with bovine cathepsin D (EC 3.4.23.5) at pH 3.2 led to the production of three polypeptides one of which, Phe⁴³-Phe⁸⁸, acted as a substrate for the carboxypeptidase. Incubation of this fragment with cathepsin A at pH 5.5 led to the release of C-terminal Phe equivalent to 50% of the total within 6h at 25°C and smaller amounts of the adjacent His (position 87) and Val (86). Purified cathepsin A was devoid of aminopeptidase and arylamidase contaminants when tested with a variety of di- and tripeptides and with the polypeptide fragment of myelin basic protein Phe⁸⁹-Arg¹⁶⁹. These studies in vitro with two different lysosomal enzymes show that cathepsin D activity is rate-limiting. A demonstration of step-wise cleavage by lysosomal enzymes may be relevant to mechanisms regulating protein turnover and to processes involved in demyelination.     

Irreversible blockade of sigma-1 receptors by haloperidol and its metabolites in guinea pig brain and SH-SY5Y human neuroblastoma cells

We evaluated the effect of haloperidol (HP) and its metabolites on [³H](+)-pentazocine binding to σ₁ receptors in SH-SY5Y human neuroblastoma cells and guinea pig brain P₁, P₂ and P₃ subcellular fractions. Three days after a single i.p. injection in guinea pigs of HP (but not of other σ₁ antagonists or (−)-sulpiride), [³H](+)-pentazocine binding to brain membranes was markedly decreased. Recovery of σ₁ receptor density to steady state after HP-induced inactivation required more than 30 days. HP-metabolite II (reduced HP, 4-(4-chlorophenyl)-α-(4-fluorophenyl)-4-hydroxy-1-piperidinebutanol), but not HP-metabolite I (4-(4-chlorophenyl)-4-hydroxypiperidine), irreversibly blocked σ₁ receptors in guinea pig brain homogenate and P₂ fraction in vitro . We found similar results in SH-SY5Y cells, which suggests that this process may also take place in humans. HP irreversibly inactivated σ₁ receptors when it was incubated with brain homogenate and SH-SY5Y cells, but not when incubated with P₂ fraction membranes, which suggests that HP is metabolized to inactivate σ₁ receptors. Menadione, an inhibitor of the ketone reductase activity that leads to the production of HP-metabolite II, completely prevented HP-induced inactivation of σ₁ receptors in brain homogenates. These results suggest that HP may irreversibly inactivate σ₁ receptors in guinea pig and human cells, probably after metabolism to reduced HP.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Stomatal response to humidity: implications for transpiration

Abstract. Transpiration rates from apple leaves are analysed in terms of the ratio of latent heat flux (λ E ) to leaf net radiation ( Q ₁) and the climatological resistance ( r_i ). Increases in stomatal resistance with increasing leaf to air vapour pressure gradient ( D ), described by an empirical model, are incorporated in the analysis. This humidity effect causes the proportion of energy dissipated as latent heat to fall as Q ₁ increases, so that leaf transpiration rates in high energy environments are likely to be similar to those in lower energy environments. Boundary layer resistance ( r _a) exerts an increasingly important effect on transpiration rates as Q ₁ increases. At constant Q ₁ stomatal closure in response to increasing D results in very small changes in leaf temperature ( T ₁) across a wide range of ambient vapour pressure deficits (δ e ); r _a is then the major factor determining T ₁. The implications of these results are discussed.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.     

EFFECTS OF ENVIRONMENTAL FACTORS ON MECHANICAL PROPERTIES OF THE CELL WALL IN RICE COLEOPTILES

Cell wall properties determined by the stress-relaxation technique were studied with coleoptiles of rice seedlings grown under different environmental conditions. The cell wall was simulated by a viscoelastic model consisting of either four or an infinite number of Maxwell components. Reciprocal of relaxation time for the first component in the former model (1/τ₁) and minimum and maximum relaxation times (T_o and T_m) in the latter, in addition to the stress/strain ratio, were parameters representing cell wall properties. Parameters changed depending on the ages and regions of the coleoptilles used and 011 the environmental conditions under which rice seedlings were grown. Effects on cell wall properties of aeration during submerged growth, excision of the coleoptile tip, and exposure to small doses of red and/or far-red light were examined. In most cases, high values of 1/τ₁ and of T_m and small values of T_o were consistent with the growth potentiality of cells, while the stress/strain ratio seemed to be a consequence of elongation growth.     

Abnormality of α1Adrenergic Receptors in the Frontal Cortex of Epileptic Rats

Abstract: We found that the binding of [³H]prazosin, a selective ligand for α₁-adrenergic recognition sites, is significantly lower in the frontal cortex of the genetically epilepsy-prone rats (GEPRs), as compared with normal Sprague-Dawley rats. Scatchard analysis reveals a decrease in the B _max of [³H]prazosin binding with no change in the apparent K _D, suggesting that there are fewer α₁-adrenergic recognition sites in the frontal cortex of the GEPR. This abnormality is associated with a reduced capacity of norepinephrine (NE) to stimulate [³H]inositol monophosphate ([³H]IP₁) formation in frontal cortex slices prelabeled with [³H]inositol. No significant differences in [³H]prazosin binding as well as NE-stimulated [³H]IP₁ formation have been observed in other brain regions including hippocampus, corpus striatum, and inferior colliculus. These results indicate that a deficit in the α₁-adrenergic receptor system in the frontal cortex may play a role in the seizure process in the GEPR.     

Role of GSK-3beta activation and alpha7 nAChRs in Abeta(1-42)-induced tau phosphorylation in PC12 cells

β-amyloid peptide 1–42 (Aβ_1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer's disease. Emerging evidence indicates that Aβ_1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ_1–42-induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ_1–42 increased phosphorylation of tau at serine-202 as detected by AT8 antibody. This Aβ_1–42-induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase-3β (GSK-3β) at tyrosine-216 (GSK-3β-pY216), which was partially inhibited by the GSK-3β inhibitor, CHIR98023. Aβ_1–42-induced tau phosphorylation and increase in GSK-3β-pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A-582941 and antagonists methyllycaconitine and α-bungarotoxin. The α7 nAChR agonist and the GSK-3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ_1–42-induced tau phosphorylation, possibly involving GSK-3β. This study provides evidence of nAChR mechanisms underlying Aβ_1–42 toxicity and tau phosphorylation, which, if translated in vivo , could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer's disease.