Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

N-terminal Protein Processing: A Comparative Proteogenomic Analysis

N-terminal methionine excision (NME) and N-terminal acetylation (NTA) are two of the most common protein post-translational modifications. NME is a universally conserved activity and a highly specific mechanism across all life forms. NTA is very common in eukaryotes but occurs rarely in prokaryotes. By analyzing data sets from yeast, mammals and bacteria (including 112 million spectra from 57 bacterial species), the largest comparative proteogenomics study to date, it is shown that previous assumptions/perceptions about the specificity and purposes of NME are not entirely correct. Although NME, through the universal enzymatic specificity of the methionine aminopeptidases, results in the removal of the initiator Met in proteins when the second residue is Gly, Ala, Ser, Cys, Thr, Pro, or Val, the comparative genomic analyses suggest that this specificity may vary modestly in some organisms. In addition, the functional role of NME may be primarily to expose Ala and Ser rather than all seven of these residues. Although any of this group provide “stabilizing” N termini in the N-end rule, and de facto leave the remaining 13 amino acid types that are classed as “destabilizing” (in higher eukaryotes) protected by the initiator Met, the conservation of NME-substrate proteins through evolution suggests that the other five are not crucially important for proteins with these residues in the second position. They are apparently merely inconsequential players (their function is not affected by NME) that become exposed because their side chains are smaller or comparable to those of Ala and Ser. The importance of exposing mainly two amino acids at the N terminus, i.e. Ala and Ser, is unclear but may be related to NTA or other post-translational modifications. In this regard, these analyses also reveal that NTA is more prevalent in some prokaryotes than previously appreciated.Although methionine is used to initiate protein synthesis for essentially all proteins, it is subsequently removed in a large percentage of cases, either by cleavage of an N-terminal “signal ” peptide (as part of cellular translocation mechanisms or precursor activations) or by the action of specific methionine aminopeptidases (MetAPs). Approximately two-thirds of the proteins in any proteome are potential substrates for the latter N-terminal methionine excision (NME),¹ and MetAPs appear in all organisms from bacteria to eukaryotes (1). The second, or P2, amino acid in protein substrates is crucially important for NME because MetAP specificity mainly depends on the nature of this residue, a selectivity that is conserved across all species (1–5). These enzymes generally excise the N-terminal Met when the second residue is Gly, Ala, Ser, Thr, Cys, Pro, or Val (3, 6, 7), which are the amino acids smallest in size (based on radius of gyration of the side chain (8)). NME is a necessary process for proper cell functioning; it is included in the minimal genome set of eubacteria (9). Eukaryotes contain two MetAPs derived from a version in bacteria (MetAP1), and another found in archea (MetAP2) (11). Just as the deletion of MetAP eubacteria is lethal, the deletion of both MetAPs in yeast is also lethal (10).In 1988, Arfin and Bradshaw (2) observed that the specificity of NME coincided with that of the N-end rule (NER) (12, 13), a ubiquitin-dependent protein degradation process that is based on the recognition of N-terminal residues. The stabilizing residues for the NER include Gly, Ala, Ser, Cys, Thr, Pro, and Val and, with the exception of Met, the destabilizing residues are all found to be in the class of P2-residues that are not substrates for the MetAPs. This suggested that NME acts to release Met from proteins whose stability is unaffected by the NER creating at the same time a second class of proteins, who have the potential for regulated turnover downstream of the cotranslational processing, when, and if, the N-terminal Met is subsequently removed by a mechanism other than the cotranslational action of the MetAPs. However, despite extensive studies, this type of programmed protein turnover (requiring downstream removal of Met) has not been demonstrated to occur. An implication of this correlation is that exposing of the stabilizing residues may also contribute to increasing their lifetime.The stabilizing residues exposed by the action of the MetAPs can be further modified. The most extensive of these reactions is N-terminal acetylation (NTA), which can occur on as much as 70–80% of the mass of the soluble protein in eukaryotes. Although the specificity of the N-acetyltransferase (NAT) responsible is not as rigid as the MetAPs, the principal substrates in the stabilizing class are usually the four smallest residues (Gly, Ala, Ser, and Thr) (6, 14). A second class of NATs can also modify the retained Met when the adjacent residues are Asp, Glu or Asn (15). The functional importance of this modification (in either case) is not known although it has been suggested that it may exert a protective effect against spurious aminopeptidase cleavages. Recently, Hwang et al. (16) have extended the NER to include Nα-acetylated termini as also destabilizing thus providing another possible function for this modification. In contrast, to date, very few instances of Nα-acetylation have been observed in bacteria. Other modifications can also occur in both eukaryotes and prokaryotes although they are generally much more limited in scope.The specificity of the MetAPs suggest an apparent connection between NME and protein degradation. However, this connection has never been examined using high-throughput mass spectrometric data or a comparative genomics approach; thus it remains unclear whether exposing these stabilizing residues contributes to increasing protein half-life and thus represents a primary purpose of NME. (The connection between NME and NER in bacteria, which has an NER with a somewhat different profile (17), is even more obscure.) Recent studies provide some examples where disruption of NME via a single-residue substitution in the P2 position causes protein degradation (18–20); however, some of these experimental results are in conflict with the NER (13). Giglione et al. (20) have shown that NME triggers degradation of D2 protein in Caenorhabditis reinhardtii in the PSII complex after replacing the second (stabilizing) Thr residue by another amino acid to prevent NME. This replacement results in early degradation of D2 and instability of the PSII complex. From this, Giglione et al. (20) postulated that NME determines protein life-span via currently unknown machinery. However, because Bachmair et al. (12) classified Met as a stabilizing residue, it is not entirely clear why substituting one stabilizing residue (Met) by another one (Gly, Ala, Ser, Cys, Thr, Pro, or Val) should affect protein stability and the substitution may have other deleterious effects that are manifested in different ways.The logic for analyzing NME and NER is shown in Fig. 1. NME exposes 7 different residues as new N termini of proteins. The natural conclusion that has become a dogma of NME is that these seven residues are exposed for a functional reason. The broad scope of NME suggests a universal reason that surpasses any particular protein''s role. In turn the comparative genomics postulate (function suggests conservation) leads to the conclusion that the seven residues should be evolutionarily conserved at position P2 of proteins. However, because only two out of the seven residues are conserved, we argue that one of the two assumptions in Fig. 1A must be incorrect and put forth the alternative logic depicted in Fig. 1B, which matches our analysis across dozens of species. According to this logic, NME accomplishes the goal of exposing Ala and Ser by exposing all residues with side chains smaller or comparable in size to Ala and Ser (G, T, V, P, and C). These residues are thus inconsequential players that are not functionally important (and are not evolutionarily conserved) at P2.Open in a separate windowFig. 1.Two alternative cases for NME function. A, NME exposes seven residues to be new N termini of proteins. Because this is presumably for some functional reason, the conventional assumption is that all seven residues must have functional importance as N termini. By the comparative genomics postulate (as defined in the text), evolutionary conservation of all seven at P2 should be observed. If all of these residues are not conserved, one of the two assumptions must be incorrect; either not all seven residues are important or the comparative genomics postulate is invalid. B, Given that the comparative genomics postulate holds, and only two of the seven residues are of functional importance as N termini, then the other five residues are inconsequential players and only these two residues should be evolutionarily conserved.In this report, we examine the connection between the specificity of NME and stabilizing residues of NER. In doing so, data sets from bacteria (including 112 million mass spectrometric spectra from 57 species), yeast, and mammals, were analyzed for N-terminal peptides both with respect to the excision (or not) of initiator Met residues and the distribution of P2-residues. The results reveal a strong preference of Ala and Ser as P2-residues. However, this process does not appear to be linked to the NER other than being generally compatible with it. These studies also demonstrate a much greater than expected number of Nα-acetylation events in some bacteria.