cAMP compartmentation is responsible for a local activation of cardiac Ca2+ channels by beta-adrenergic agonists.

The role of cAMP subcellular compartmentation in the progress of beta-adrenergic stimulation of cardiac L-type calcium current (ICa) was investigated by using a method based on the use of whole-cell patch-clamp recording and a double capillary for extracellular microperfusion. Frog ventricular cells were sealed at both ends to two patch-clamp pipettes and positioned approximately halfway between the mouths of two capillaries that were separated by a 5-micron thin wall. ICa could be inhibited in one half or the other by omitting Ca2+ from one solution or the other. Exposing half of the cell to a saturating concentration of isoprenaline (ISO, 1 microM) produced a nonmaximal increase in ICa (347 +/- 70%; n = 4) since a subsequent application of ISO to the other part induced an additional effect of nearly similar amplitude to reach a 673 +/- 130% increase. However, half-cell exposure to forskolin (FSK, 30 microM) induced a maximal stimulation of ICa (561 +/- 55%; n = 4). This effect was not the result of adenylyl cyclase activation due to FSK diffusion in the nonexposed part of the cell. To determine the distant effects of ISO and FSK on ICa, the drugs were applied in a zero-Ca solution. Adding Ca2+ to the drug-containing solutions allowed us to record the local effect of the drugs. Dose-response curves for the local and distant effects of ISO and FSK on ICa were used as an index of cAMP concentration changes near the sarcolemma. We found that ISO induced a 40-fold, but FSK induced only a 4-fold, higher cAMP concentration close to the Ca2+ channels, in the part of the cell exposed to the drugs, than it did in the rest of the cell. cAMP compartmentation was greatly reduced after inhibition of phosphodiesterase activity with 3-isobutyl-methylxanthine, suggesting the colocalization of enzymes involved in the cAMP cascade. We conclude that beta-adrenergic receptors are functionally coupled to nearby Ca2+ channels via local elevations of cAMP.

Engineering an intracellular pathway for major histocompatibility complex class II presentation of antigens.

The presentation of antigenic peptides by major histocompatibility complex (MHC) class II molecules to CD4+ T cells is critical to the function of the immune system. In this study, we have utilized the sorting signal of the lysosomal-associated membrane protein LAMP-1 to target a model antigen, human papillomavirus 16 E7 (HPV-16 E7), into the endosomal and lysosomal compartments. The LAMP-1 sorting signal reroutes the antigen into the MHC class II processing pathway, resulting in enhanced presentation to CD4+ cells in vitro. In vivo immunization experiments in mice demonstrated that vaccinia containing the chimeric E7/LAMP-1 gene generated greater E7-specific lymphoproliferative activity, antibody titers, and cytotoxic T-lymphocyte activities than vaccinia containing the wild-type HPV-16 E7 gene. These results suggest that specific targeting of an antigen to the endosomal and lysosomal compartments enhances MHC class II presentation and vaccine potency.

Cytoplasmic free-Ca2+ level rises with repellents and falls with attractants in Escherichia coli chemotaxis.

Cytoplasmic free-Ca2+ levels in Escherichia coli were measured by use of the fluorescent Ca(2+)-indicator dye fura-2. Chemotactically wild-type E. coli regulated cytoplasmic free Ca2+ at approximately 100 nM when no stimuli were encountered, but changes in bacterial behavior correlated with changes in cytoplasmic free-Ca2+ concentration. For chemotactically wild-type E. coli, addition of a repellent resulted in cells tumbling and a transient increase in cytoplasmic free-Ca2+ levels. Conversely, addition of an attractant to wild-type cells caused running and produced a transient decrease in cytoplasmic free-Ca2+ levels. Studies with mutant strains showed that the chemoreceptors were required for the observed changes in cytoplasmic free-Ca2+ levels in response to chemical stimuli.

Detection of Ca2+ entry through mechanosensitive channels localizes the site of mechanoelectrical transduction in hair cells.

A hair cell, the sensory receptor of the internal ear, transduces mechanical stimuli into electrical responses. Transduction results from displacement of the hair bundle, a cluster of rod-shaped stereocilia extending from the cell's apical surface. Biophysical experiments indicate that, by producing shear between abutting stereocilia, a bundle displacement directly opens cation-selective transduction channels. Specific models of gating depend on the location of these channels, which has been controversial: although some physiological and immunocytochemical experiments have situated the transduction channels at the hair bundle's top, monitoring of fluorescence signals from the Ca2+ indicator fura-2 has instead suggested that Ca2+ traverses channels at the bundle's base. To examine the site of Ca2+ entry through transduction channels, we used laser-scanning confocal microscopy, with a spatial resolution of < 1 micron and a temporal resolution of < 2 ms, to observe hair cells filled with the indicator fluo-3. An unstimulated hair cell showed a "tip blush" of enhanced fluorescence at the hair bundle's top, which we attribute to Ca2+ permeation through transduction channels open at rest. Upon mechanical stimulation, individual stereocilia displayed increased fluorescence that originated near their tips, then spread toward their bases. Our results confirm that mechanoelectrical transduction occurs near stereociliary tips.

Intracellular coexistence of methano- and thioautotrophic bacteria in a hydrothermal vent mussel.

The coexistence of two phylogenetically distinct symbiont species within a single cell, a condition not previously known in any metazoan, is demonstrated in the gills of a Mid-Atlantic Ridge hydrothermal vent mussel (family Mytilidae). Large and small symbiont morphotypes within the gill bacteriocytes are shown to be separate bacterial species by molecular phylogenetic analysis and fluorescent in situ hybridization. The two symbiont species are affiliated with thioautotrophic and methanotrophic symbionts previously found in monospecific associations with closely related mytilids from deep-sea hydrothermal vents and hydrocarbon seeps.

Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.

Simultaneous measurements of cytosolic free Ca2+ concentration and insulin release, in mouse single pancreatic islets, revealed a direct correlation only initially after stimulation with glucose or K+. Later, there is an apparent dissociation between these two parameters, with translocation of alpha and epsilon isoenzymes of protein kinase C to membranes and simultaneous desensitization of insulin release in response to glucose. Recovery of insulin release, without any concomitant changes in cytosolic free Ca2+ concentration, after addition of phorbol 12-myristate 13-acetate, okadaic acid, and forskolin supports the notion that the desensitization process is accounted for by dephosphorylation of key regulatory sites of the insulin exocytotic machinery.

Molecular and biochemical characterization of dNOS: a Drosophila Ca2+/calmodulin-dependent nitric oxide synthase.

Nitric oxide (NO) is an intercellular messenger involved with various aspects of mammalian physiology ranging from vasodilation and macrophage cytotoxicity to neuronal transmission. NO is synthesized from L-arginine by NO synthase (NOS). Here, we report the cloning of a Drosophila NOS gene, dNOS, located at cytological position 32B. The dNOS cDNA encodes a protein of 152 kDa, with 43% amino acid sequence identity to rat neuronal NOS. Like mammalian NOSs, DNOS protein contains putative binding sites for calmodulin, FMN, FAD, and NADPH. DNOS activity is Ca2+/calmodulin dependent when expressed in cell culture. An alternative RNA splicing pattern also exists for dNOS, which is identical to that for vertebrate neuronal NOS. These structural and functional observations demonstrate remarkable conservation of NOS between vertebrates and invertebrates.

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.

A trace component of ginseng that inhibits Ca2+ channels through a pertussis toxin-sensitive G protein.

A crude extract from ginseng root inhibits high-threshold, voltage-dependent Ca2+ channels through an unknown receptor linked to a pertussis toxin-sensitive G protein. We now have found the particular compound that seems responsible for the effect: it is a saponin, called ginsenoside Rf (Rf), that is present in only trace amounts within ginseng. At saturating concentrations, Rf rapidly and reversibly inhibits N-type, and other high-threshold, Ca2+ channels in rat sensory neurons to the same degree as a maximal dose of opioids. The effect is dose-dependent (half-maximal inhibition: 40 microM) and it is virtually eliminated by pretreatment of the neurons with pertussis toxin, an inhibitor of G(o) and Gi GTP-binding proteins. Other ginseng saponins--ginsenosides Rb1, Rc, Re, and Rg1--caused relatively little inhibition of Ca2+ channels, and lipophilic components of ginseng root had no effect. Antagonists of a variety of neurotransmitter receptors that inhibit Ca2+ channels fail to alter the effect of Rf, raising the possibility that Rf acts through another G protein-linked receptor. Rf also inhibits Ca2+ channels in the hybrid F-11 cell line, which might, therefore, be useful for molecular characterization of the putative receptor for Rf. Because it is not a peptide and it shares important cellular and molecular targets with opioids, Rf might be useful in itself or as a template for designing additional modulators of neuronal Ca2+ channels.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Inhibition of selective signaling events in natural killer cells recognizing major histocompatibility complex class I.

Many studies have characterized the transmembrane signaling events initiated after T-cell antigen receptor recognition of major histocompatibility complex (MHC)-bound peptides. Yet, little is known about signal transduction from a set of MHC class I recognizing receptors on natural killer (NK) cells whose ligation dramatically inhibits NK cell-mediated killing. In this study we evaluated the influence of MHC recognition on the proximal signaling events in NK cells binding tumor targets. We utilized two experimental models where NK cell-mediated cytotoxicity was fully inhibited by the recognition of specific MHC class I molecules. NK cell binding to either class I-deficient or class I-transfected target cells initiated rapid protein tyrosine kinase activation. In contrast, whereas NK cell binding to class I-deficient targets led to inositol phosphate release and increased intracellular free calcium ([Ca2+]i), NK recognition of class I-bearing targets did not induce the activation of these phospholipase C-dependent signaling events. The recognition of class I by NK cells clearly had a negative regulatory effect since blocking this interaction using anti-class I F(ab')2 fragments increased inositol 1,4,5-trisphosphate release and [Ca2+]i and increased the lysis of the targets. These results suggest that one of the mechanisms by which NK cell recognition of specific MHC class I molecules can block the development of cell-mediated cytotoxicity is by inhibiting specific critical signaling events.

Sphingosylphosphocholine, a signaling molecule which accumulates in Niemann-Pick disease type A, stimulates DNA-binding activity of the transcription activator protein AP-1.

Sphingosylphosphocholine (SPC) is the deacylated derivative of sphingomyelin known to accumulate in neuropathic Niemann-Pick disease type A. SPC is a potent mitogen that increases intracellular free Ca2+ and free arachidonate through pathways that are only partly protein kinase C-dependent. Here we show that SPC increased specific DNA-binding activity of transcription activator AP-1 in electrophoretic mobility-shift assays. Increased DNA-binding activity of AP-1 was detected after only 1-3 min, was maximal after 6 hr, and remained elevated at 12-24 hr. c-Fos was found to be a component of the AP-1 complex. Northern hybridization revealed an increase in c-fos transcripts after 30 min. Since the increase in AP-1 binding activity preceded the increase in c-fos mRNA, posttranslational modifications may be important in mediating the early SPC-induced increases in AP-1 DNA-binding activity. Western analysis detected increases in nuclear c-Jun and c-Fos proteins following SPC treatment. SPC also transactivated a reporter gene construct through the AP-1 recognition site, indicating that SPC can regulate the expression of target genes. Thus, SPC-induced cell proliferation may result from activation of AP-1, linking signal transduction by SPC to gene expression. Since the expression of many proteins with diverse functions is known to be regulated by AP-1, SPC-induced activation of AP-1 may contribute to the pathophysiology of Niemann-Pick disease.

A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids.

The synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], the immediate precursor of intracellular signals generated by calcium-mobilizing hormones and growth factors, is initiated by the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate [PtdIns(4)P] by phosphatidylinositol 4-kinase (PtdIns 4-kinase). Although cells contain several PtdIns 4-kinases, the enzyme responsible for regulating the synthesis of hormone-sensitive PtdIns(4,5)P2 pools has not been identified. In this report we describe the inhibitory effect of micromolar concentrations of wortmannin (WT) on the synthesis of hormone-sensitive PtdIns(4)P and PtdIns(4,5)P2 pools in intact adrenal glomerulosa cells, and the presence of a WT-sensitive PtdIns 4-kinase in adrenocortical extracts. In addition to its sensitivity to the PtdIns 3-kinase inhibitor WT, this enzyme is distinguished from the recognized membrane-bound PtdIns 4-kinases by its molecular size and weak membrane association. Inhibition of this PtdIns 4-kinase by WT results in rapid loss of the hormone-sensitive PtdIns(4,5)P2 pool in angiotensin II-stimulated glomerulosa cells. Consequently, WT treatment inhibits the sustained but not the initial increases in inositol 1,4,5-trisphosphate and cytoplasmic [Ca2+] in a variety of agonist-stimulated cells, including adrenal glomerulosa cells, NIH 3T3 fibroblasts, and Jurkat lymphoblasts. These results indicate that a specific WT-sensitive PtdIns 4-kinase is critical for the maintenance of the agonist-sensitive polyphosphoinositide pool in several cell types.

Induction of intracellular cAMP by a synthetic retroviral envelope peptide: a possible mechanism of immunopathogenesis in retroviral infections.

A synthetic heptadecapeptide, CKS-17, represents the highly conserved amino acid sequences occurring within the transmembrane envelope protein of many animal and human retroviruses. CKS-17 has been demonstrated to exhibit suppressive properties for numerous immune functions. We have recently shown that CKS-17 acts as an immunomodulatory epitope causing an imbalance of human type 1 and type 2 cytokine production and suppression of cell-mediated immunities. cAMP, an intracellular second messenger, plays an important role in regulation of cytokine biosynthesis--i.e., elevation of intracellular cAMP levels selectively inhibits type 1 cytokine production but has no effect or enhances type 2 cytokine production. Here, we demonstrate that CKS-17 induces dramatic rises in the intracellular cAMP levels of a human monocyte cell line and of human peripheral blood mononuclear cells in a time- and dose-dependent manner. A peptide corresponding to the reverse sequence of CKS-17, used as control, has no effect on intracellular cAMP levels. The cAMP-inducing ability of CKS-17 is significantly blocked by SQ-22536, an inhibitor of adenylate cyclase. These results indicate that CKS-17, a highly conserved component of the transmembrane proteins of immunosuppressive retroviruses, induces increased intracellular levels of cAMP via activation of adenylate cyclase and suggest that this retroviral envelope peptide may differentially modulate type 1 and type 2 cytokine production through elevation of intracellular cAMP levels.