Pain is unique among sensations in that the perceived intensity increases, or sensitizes, during exposure to a strong stimulus. One important mediator of sensitization is bradykinin (BK), a peptide released as a consequence of tissue damage. BK enhances the membrane ionic current activated by heat in nociceptive neurons, using a pathway that involves activation of protein kinase C (PKC). We find that five PKC isoforms are present in sensory neurons but that only PKC-epsilon is translocated to the cell membrane by BK. The heat response is sensitized when constitutively active PKC-epsilon is incorporated into nociceptive neurons. Conversely, BK-induced sensitization is suppressed by a specific peptide inhibitor of PKC-epsilon. We conclude that PKC-epsilon is principally responsible for sensitization of the heat response in nociceptors by bradykinin.
Bacteria have developed many fascinating antibiotic-resistance mechanisms. A protein in Lactococcus lactis, LmrA, mediates antibiotic resistance by extruding amphiphilic compounds from the inner leaflet of the cytoplasmic membrane. Unlike other known bacterial multidrug-resistance proteins, LmrA is an ATP-binding cassette (ABC) transporter. The human multidrug-resistance P-glycoprotein, encoded by the MDR1 gene, is also an ABC transporter, overexpression of which is one of the principal causes of resistance of human cancers to chemotherapy. We expressed lmrA in human lung fibroblast cells. Surprisingly, LmrA was targeted to the plasma membrane and conferred typical multidrug resistance on these human cells. The pharmacological characteristics of LmrA and P-glycoprotein-expressing lung fibroblasts were very similar, and the affinities of both proteins for vinblastine and magnesium-ATP were indistinguishable. Blockers of P-glycoprotein-mediated multidrug resistance also inhibited LmrA-dependent drug resistance. Kinetic analysis of drug dissociation from LmrA expressed in plasma membranes of insect cells revealed the presence of two allosterically linked drug-binding sites indistinguishable from those of P-glycoprotein. These findings have implications for the reversal of antibiotic resistance in pathogenic microorganisms. Taken together, they demonstrate that bacterial LmrA and human P-glycoprotein are functionally interchangeable and that this type of multidrug-resistance efflux pump is conserved from bacteria to man.
Multidrug resistance in acute myeloid leukemia is often conferred by overexpression of P-glycoprotein, encoded by the MDR1 gene. We have characterized the key regulatory steps in the development of multidrug resistance in K562 myelogenous leukemic cells. Unexpectedly, up-regulation of MDR1 levels was not due to transcriptional activation but was achieved at two distinct posttranscriptional steps, mRNA turnover and translational regulation. The short-lived (half-life 1 h) MDR1 mRNA of naïve cells (not exposed to drugs) was stabilized (halflife greater than 10 h) following short-term drug exposure. However, this stabilized mRNA was not associated with translating polyribosomes and did not direct Pglycoprotein synthesis. Selection for drug resistance, by long-term exposure to drug, led to resistant lines in which the translational block was overcome such that the stabilized mRNA was translated and P-glycoprotein expressed. The absence of a correlation between steadystate MDR1 mRNA and P-glycoprotein levels was not restricted to K562 cells but was found in other lymphoid cell lines. These findings have implications for the avoidance or reversal of multidrug resistance in the clinic. MDR1 is the most common impediment to successful chemotherapy for a variety of cancers (1). The most frequent form of drug resistance in relapsed acute leukemia is overexpression of P-glycoprotein (2, 3). P-glycoprotein is a member of the ATPbinding cassette superfamily of active transporters and functions as an energy-dependent efflux pump that reduces the intracellular concentration of cytotoxic compounds and, hence, their toxicity. P-glycoprotein has a broad substrate specificity and can confer resistance to a wide range of different cytotoxic compounds (4).Most pre-clinical and clinical efforts to overcome MDR aim to modulate P-glycoprotein activity. However, clinical trials of compounds that inhibit P-glycoprotein activity have had limited success and led to adverse pharmacokinetic side effects (1). It may, therefore, be more appropriate to target MDR1 expression. Indeed, MDR1 transcription has been targeted with Ecteinascidin 743 in pre-clinical studies (5) and more recently by modulation of the nuclear receptor SXR (6). Strategies involving antisense and transcriptional decoy (7) and the use of anti-MDR1 mRNA hammerhead ribozymes have also been suggested (8).Stresses such as short-term exposure to cytotoxic drugs results in the up-regulation of MDR1 mRNA levels in many cell lines (9 -13) and in human metastatic sarcomas in vivo (14). This is frequently due to transcriptional activation of the MDR1 gene and has been reported in many cell lines after different physical and chemical stimulations and in cells selected for resistance to a variety of cytotoxic drugs (5,9,10,15,16). In cell lines selected for drug resistance, increased MDR1 gene expression is also the result of amplification of the MDR1 locus and the appearance of self-replicating episomes (4). Gene rearrangements that constitutively activate MDR1 transcription have also...
Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated.
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