Mitochondrial carbonic anhydrase CA VB: Differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles

A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human–mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

Intracellular polyamines mediate inward rectification of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that lack the glutamate receptor GluR2 subunit are Ca(2+)-permeable and exhibit inwardly rectifying current responses to kainate and AMPA. A proportion of cultured rat hippocampal neurons show similar Ca(2+)-permeable inwardly rectifying AMPA receptor currents. Inward rectification in these neurons was lost with intracellular dialysis and was not present in excised outside-out patches but was maintained in perforated-patch whole-cell recordings, suggesting that a diffusible cytoplasmic factor may be responsible for rectification. Inclusion of the naturally occurring polyamines spermine and spermidine in the recording pipette prevented loss of rectification in both whole-cell and excised-patch recordings; Mg2+ and putrescine were without effect. Inward rectification of Ca(2+)-permeable AMPA receptors may reflect voltage-dependent channel block by intracellular polyamines.

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Slow and Prolonged Activation of the p47 Protein Kinase during Hypersensitive Cell Death in a Culture of Tobacco Cells

To investigate the involvement of protein kinases in the signaling cascade that leads to hypersensitive cell death, we used a previously established system in which a fungal elicitor, xylanase from Trichoderma viride (TvX), induces a hypersensitive reaction in tobacco (Nicotiana tabacum) cells in culture (line XD6S). The elicitor induced the slow and prolonged activation of a p47 protein kinase, which has the characteristics of a family member of the mitogen-activated protein kinases. An inhibitor of protein kinases, staurosporine, and a blocker of Ca channels, Gd3+ ions, both of which blocked the TvX-induced hypersensitive cell death, inhibited the TvX-induced activation of p47 protein kinase. Moreover, an inhibitor of serine/threonine protein phosphatase alone induced both rapid cell death and the persistent activation of the p47 protein kinase. Thus, the p47 protein kinase might be a component of the signal transduction pathway that leads to hypersensitive cell death, and the regulation of the duration of activation of the p47 protein kinase might be important in determining the destiny of tobacco cells.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.

Inhibition of NF-AT-dependent transcription by NF-kappa B: implications for differential gene expression in T helper cell subsets.

Activation of individual CD4+ T cells results in differential lymphokine expression: interleukin 2 (IL-2) is preferentially produced by T helper type 1 (TH1) cells, which are involved in cell-mediated immune responses, whereas IL-4 is synthesized by TH2 cells, which are essential for humoral immunity. The Ca(2+)-dependent factor NF-ATp plays a key role in the inducible transcription of both these lymphokine genes. However, while IL2 expression requires the contribution of Ca(2+)- and protein kinase C-dependent signals, we report that activation of human IL4 transcription through the Ca(2+)-dependent pathway is diminished by protein kinase C stimulation in Jurkat T cells. This phenomenon is due to mutually exclusive binding of NF-ATp and NF-kappa B to the P sequence, an element located 69 bp upstream of the IL4 transcription initiation site. Human IL4 promoter-mediated transcription is downregulated in Jurkat cells stimulated with the NF-kappa B-activating cytokine tumor necrosis factor alpha and suppressed in RelA-overexpressing cells. In contrast, protein kinase C stimulation or RelA overexpression does not affect the activity of a human IL4 promoter containing a mouse P sequence, which is a higher-affinity site for NF-ATp and a lower-affinity site for RelA. Thus, competition between two general transcriptional activators, RelA and NF-ATp, mediates the inhibitory effect of protein kinase C stimulation on IL4 expression and may contribute to differential gene expression in TH cells.

Human immunodeficiency virus reverse transcriptase substrate-induced conformational changes and the mechanism of inhibition by nonnucleoside inhibitors.

A combination of transient kinetic and equilibrium titration methods has been used to show that both primer/template and nucleotide binding to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase are two-step processes. In both cases, after initial formation of relatively weakly bound states, isomerization reactions lead to tightly bound states. In the case of deoxynucleotide binding to the reverse transcriptase-primer/template complex, the second step in the interaction is rate-limiting in the overall reaction during processive polymerization. Discrimination against incorrect nucleotides occurs both in the initial weak binding and in the second step but is purely kinetic in the second step (as opposed to thermodynamic in the first step). Nonnucleoside inhibitors have a relatively small effect on nucleotide-binding steps (overall affinity is reduced by a factor of ca. 10), while the affinity of the primer/template duplex is increased by at least a factor of 10. The major effect of nonnucleoside inhibitors is on the chemical step (nucleotide transfer).

Retro and retroenantio analogs of cecropin-melittin hybrids.

Hybrid analogs of cecropin A (CA) and melittin (M), which are potent antibacterial peptides, have been synthesized. To understand the structural requirements for this antibacterial activity, we have also synthesized the enantio, retro, and retroenantio isomers of two of the hybrids and their N-terminally acetylated derivatives. All analogs of CA(1-13)M(1-13)-NH2 were as active as the parent peptide against five test bacterial strains, but one bacterial strain was resistant to the retro and retroenantio derivatives. Similarly, all analogs of CA(1-7)M(2-9)-NH2 were active against four strains, while two strains were resistant to the retro and retroenantio analogs containing free NH3+ end groups, but acetylation restored activity against one of them. From these data it was concluded that chirality of the peptide was not a critical feature, and full activity could be achieved with peptides containing either all L- or all D-amino acids in their respective right-handed or left-handed helical conformations. For most of the bacterial strains, the sequence of these peptides or the direction of the peptide bonds could be critical but not both at the same time. For some strains, both needed to be conserved.