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PubMed1986

The alteration of myosin isoform compartmentation in specific mutants of Caenorhabditis elegans.

Myosin isoforms A and B are located at the surface of the central and polar regions, respectively, of thick filaments in body muscle cells of Caenorhabditis elegans, whereas paramyosin and a distinct core structure comprise the backbones of these filaments. Thick filaments and related structures were isolated from nematode mutants that have altered thick filament protein compositions. These mutant filaments and their complexes with specific antibodies were studied by electron microscopy to determine the distribution of the two myosins. The compartmentation of the two myosin isoforms in body wall muscle thick filaments depends not only upon the intrinsic properties of the myosins but their interactions with other components such as paramyosin and their relative quantities determined by synthesis.

Animals↗
PubMed1992

Compartmentalization of PDGF on extracellular binding sites dependent on exon-6-encoded sequences.

The PDGFs are a family of molecules assembled as disulfide-bonded homo- and heterodimers from two distinct but highly homologous polypeptide chains (PDGF-A and PDGF-B). Two PDGF A-chain transcripts, which arise from alternative usage of the 69-bp exon 6 and exon 7, give rise to two forms of PDGF-A. In spite of the conservation of two PDGF A-chain forms over at least 350 million years, no differences in their biological activities have been identified. We have investigated the activity of the sequence encoded by the alternatively spliced exon 6 of the PDGF A-chain (peptide AL). Addition of peptide AL at 10(-5)-10(-9) M to cultured endothelium and smooth muscle induced a dose-dependent, 3-20-fold increase in PDGF in conditioned media within 30 min. Peptide AL had no detectable effect on A- or B-chain transcript levels, and decrease in culture temperature did not prevent rapid release of PDGF. In human umbilical vein endothelial cells treated with peptide AL, the PDGF release was principally PDGF-BB, while in smooth muscle cells it was primarily PDGF-AA. The capacity to induce release of PDGF is shared by the homologous peptide encoded by exon 6 of the B-chain of PDGF. Binding studies and cross-linking analysis are consistent with a charge-based association of exon 6 sequences with membrane- and matrix-associated heparan-sulfate proteoglycans. We hypothesize that translation of exon 6 of the A- or B-chain of PDGF results in compartmentalization of these forms of PDGF with HS-PG, whereas forms lacking this sequence would be soluble and diffuse.

Amino Acid Sequence↗
PubMed1994

Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis.

Movements of transferrin and alpha 2-macroglobulin receptor molecules in the plasma membrane of cultured normal rat kidney (NRK) fibroblastic cells were investigated by video-enhanced contrast optical microscopy with 1.8 nm spatial precision and 33 ms temporal resolution by labeling the receptors with the ligand-coated nanometer-sized colloidal gold particles. For both receptor species, most of the movement trajectories are of the confined diffusion type, within domains of approximately 0.25 microns2 (500-700 nm in diagonal length). Movement within the domains is random with a diffusion coefficient approximately 10(-9) cm2/s, which is consistent with that expected for free Brownian diffusion of proteins in the plasma membrane. The receptor molecules move from one domain to one of the adjacent domains at an average frequency of 0.034 s-1 (the residence time within a domain approximately 29 s), indicating that the plasma membrane is compartmentalized for diffusion of membrane receptors and that long-range diffusion is the result of successive intercompartmental jumps. The macroscopic diffusion coefficients for these two receptor molecules calculated on the basis of the compartment size and the intercompartmental jump rate are approximately 2.4 x 10(-11) cm2/s, which is consistent with those determined by averaging the long-term movements of many particles. Partial destruction of the cytoskeleton decreased the confined diffusion mode, increased the simple diffusion mode, and induced the directed diffusion (transport) mode. These results suggest that the boundaries between compartments are made of dynamically fluctuating membrane skeletons (membrane-skeleton fence model).

Animals↗
PubMed2002

The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a phagosome and grows in the host cell cytosol. The pore-forming cholesterol-dependent cytolysin, listeriolysin O (LLO), mediates bacterial escape from vesicles and is approximately 10-fold more active at an acidic than neutral pH. By swapping dissimilar residues from a pH-insensitive orthologue, perfringolysin O (PFO), we identified leucine 461 as unique to pathogenic Listeria and responsible for the acidic pH optimum of LLO. Conversion of leucine 461 to the threonine present in PFO increased the hemolytic activity of LLO almost 10-fold at a neutral pH. L. monocytogenes synthesizing LLO L461T, expressed from its endogenous site on the bacterial chromosome, resulted in a 100-fold virulence defect in the mouse listeriosis model. These bacteria escaped from acidic phagosomes and initially grew normally in cells and spread cell to cell, but prematurely permeabilized the host membrane and killed the cell. These data show that the acidic pH optimum of LLO results from an adaptive mutation that acts to limit cytolytic activity to acidic vesicles and prevent damage in the host cytosol, a strategy also used by host cells to compartmentalize lysosomal hydrolases.

Acids↗
PubMed2003

Compartmentalization of integrin alpha6beta4 signaling in lipid rafts.

Integrin alpha6beta4 signaling proceeds through Src family kinase (SFK)-mediated phosphorylation of the cytoplasmic tail of beta4, recruitment of Shc, and activation of Ras and phosphoinositide-3 kinase. Upon cessation of signaling, alpha6beta4 mediates assembly of hemidesmosomes. Here, we report that part of alpha6beta4 is incorporated in lipid rafts. Metabolic labeling in combination with mutagenesis indicates that one or more cysteine in the membrane-proximal segment of beta4 tail is palmitoylated. Mutation of these cysteines suppresses incorporation of alpha6beta4 in lipid rafts, but does not affect alpha6beta4-mediated adhesion or assembly of hemidesmosomes. The fraction of alpha6beta4 localized to rafts associates with a palmitoylated SFK, whereas the remainder does not. Ligation of palmitoylation-defective alpha6beta4 does not activate SFK signaling to extracellular signal-regulated kinase and fails to promote keratinocyte proliferation in response to EGF. Thus, compartmentalization in lipid rafts is necessary to couple the alpha6beta4 integrin to a palmitoylated SFK and promote EGF-dependent mitogenesis.

Amino Acid Sequence↗
PubMed2006

The secretory membrane system in the Drosophila syncytial blastoderm embryo exists as functionally compartmentalized units around individual nuclei.

Drosophila melanogaster embryogenesis begins with 13 nuclear division cycles within a syncytium. This produces >6,000 nuclei that, during the next division cycle, become encased in plasma membrane in the process known as cellularization. In this study, we investigate how the secretory membrane system becomes equally apportioned among the thousands of syncytial nuclei in preparation for cellularization. Upon nuclear arrival at the cortex, the endoplasmic reticulum (ER) and Golgi were found to segregate among nuclei, with each nucleus becoming surrounded by a single ER/Golgi membrane system separate from adjacent ones. The nuclear-associated units of ER and Golgi across the syncytial blastoderm produced secretory products that were delivered to the plasma membrane in a spatially restricted fashion across the embryo. This occurred in the absence of plasma membrane boundaries between nuclei and was dependent on centrosome-derived microtubules. The emergence of secretory membranes that compartmentalized around individual nuclei in the syncytial blastoderm is likely to ensure that secretory organelles are equivalently partitioned among nuclei at cellularization and could play an important role in the establishment of localized gene and protein expression patterns within the early embryo.

Animals↗
PubMed1981

Subcellular compartmentalization of maize storage proteins in Xenopus oocytes injected with zein messenger RNAs.

Maize storage proteins synthesized in oocytes were compartmentalized in membrane vesicles because they were resistant to hydrolysis by protease, unless detergent was present. The site of storage protein deposition within the oocyte was determined by subcellular fractionation. Optimal separation of oocyte membranes and organelles was obtained when EDTA and high concentrations of NaCl were included in the homogenization and gradient buffers. Under these conditions, fractions in sucrose gradients containing a heterogeneous mixture of smooth membranes (presumably endoplasmic reticulum, Golgi apparatus, and plasma membrane, density = 1.10-1.12 g/cm3), mitochondria (densities = 1.14 and 1.16 g/cm3), yolk platelets (density = 1.21 g/cm3), and a dense matrix material (density = 1.22 g/cm3) could be separated. Some zein proteins were recovered in the mixed membrane fraction, but the majority occurred in vesicles sedimenting with yolk platelets and granular material at a density of approximately 1.22 g/cm3. When metrizamide was included in the gradient to increase the density, little of the dense matrix material was isolated, and vesicles containing zein proteins were separated from other oocyte components. These vesicles were similar to protein bodies in maize endosperm because they were of identical density and contained the same group of polypeptides.

Animals↗
PubMed1971

The compartmentalization of antigen-reactive lymphocytes in desensitized guinea pigs.

A single injection of epsilon,DNP-Lys(7-10) can render previously sensitized guinea pigs specifically unreactive to subsequent intradermal challenge with that antigen. Antigen-reactive lymphocytes, as assayed by macrophage-migration in-inhibition or thymidine incorporation, were depleted from the peritoneal exudates of those animals. In contrast, it was intriguing to find that lymph node lymphocytes from such animals responded normally in the antigen-induced thymidine incorporation assay. These studies demonstrate a compartmentalization of antigen-reactive lymphocytes in desensitized animals which may account for the short-lived nature of this phenomenon.

Animals↗
PubMed1996

Mechanisms of platelet-activating factor-induced lipid body formation: requisite roles for 5-lipoxygenase and de novo protein synthesis in the compartmentalization of neutrophil lipids.

Lipid bodies, lipid rich cytoplasmic inclusions, are characteristically abundant in vivo in leukocytes associated with inflammation. Because lipid bodies are potential reservoirs of esterified arachidonate and sites at which eicosanoid-forming enzymes may localize, we evaluated mechanisms of lipid body formation in neutrophils (PMN). Among receptor-mediated agonists, platelet activating factor (PAF), but not C5a, formyl-methyl-phenylalanine, interleukin 8, or leukotriene (LT) B4, induced the rapid formation of lipid bodies in PMN. This action of PAF was receptor mediated, as it was dose dependently inhibited by the PAF receptor antagonist WEB 2086 and blocked by pertussis toxin. Lipid body induction by PAF required 5-lipoxygenase (LO) activity and was inhibited by the 5-lipoxygenase-activating protein antagonist MK 886 and the 5-LO inhibitor zileuton, but not by cyclooxygenase inhibitors. Corroborating the dependency of PAF-induced lipid body formation on 5-LO, PMN and macrophages from wild-type mice, but not from 5-LO genetically deficient mice, formed lipid bodies on exposure to PAF both in vitro and in vivo within the pleural cavity. The 5-LO product inducing lipid body formation was not LTB4 but was 5(S)-hydroxyeicosatetraenoic acid [5(S)-HETE], which was active at 10-fold lower concentrations than PAF and was also inhibited by pertussis toxin but not by zileuton or WEB 2086. Furthermore, 5-HETE was equally effective in inducing lipid body formation in both wild-type and 5-LO genetically deficient mice. Both PAF- and 5(S)-HETE-induced lipid body formation were inhibited by protein kinase C (PKC) inhibitors staurosporine and chelerythrine, the phospholipase C (PLC) inhibitors D609 and U-73122, and by actinomycin D and cycloheximide. Prior stimulation of human PMN with PAF to form lipid bodies enhanced eicosanoid production in response to submaximal stimulation with the calcium ionophore A23187; and the levels of both prostaglandin (PG) E2 and LTB4 correlated with the number of lipid bodies. Furthermore, pretreatment of cells with actinomycin D or cycloheximide inhibited not only the induction of lipid body formation by PAF, but also the PAF-induced "priming" for enhanced PGE2 and LTB4 in PMN. Thus, the compartmentalization of lipids to form lipid bodies in PMN is dependent on specific cellular responses that can be PAF receptor mediated, involves signaling through 5-LO to form 5-HETE and then through PKC and PLC, and requires new protein synthesis. Since increases in lipid body numbers correlated with priming for enhanced PGE2 and LTB4 production in PMN, the induction of lipid bodies may have a role in the formation of eicosanoid mediators by leukocytes involved in inflammation.

Animals↗
PubMed2000

Human immunodeficiency virus type 1 shedding pattern in semen correlates with the compartmentalization of viral Quasi species between blood and semen.

High levels of human immunodeficiency virus (HIV) type 1 have been detected in semen at all stages of disease. However, it is not clear whether HIV-1 is shed in semen continuously or intermittently. In a prospective longitudinal study, viral RNA was measured weekly for 10 weeks in semen and blood of HIV-seropositive subjects. Results showed three different patterns of HIV-1 shedding in semen: none (28%), continuous (28%), and intermittent (44%). In contrast, there was no change in blood plasma virus load during the study period. Phylogenetic analysis of the envelope sequences of HIV-1 RNA in semen and blood revealed distinct virus populations in semen and blood of intermittent shedders but similar virus populations in the semen and blood of continuous shedder. These results indicate for the first time that HIV-1 is shed primarily in an intermittent manner and that shedding patterns of HIV-1 in semen are related to compartmentalization of HIV-1 between semen and blood.

Cell Compartmentation↗
PubMed2003

Compartmentalized nitrosation and nitration in mitochondria.

A wide spectrum of the biological actions of nitric oxide and its oxidizing metabolites are mediated via mitochondria. Mitochondria are highly compartmentalized organelles consisting of three distinct compartments: the matrix, the intermembrane space, and the membranes. These compartments are different in their electrochemical properties, redox state, pH, enzymes, and ion content. Nitric oxide and its reactive species react within these compartments in distinct manners. The mitochondrial intermembrane space provides an environment that favors S-nitrosation, whereas nitration occurs largely within the matrix. This article will review some of the interactions of these species with certain mitochondrial respiratory chain complexes, apoptotic proteins, and enzymes. The reversibility and the suborganelle preference of these reactions will be discussed.

Animals↗
PubMed1992

HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.

Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.

Base Sequence↗
PubMed1997

Compartmentation of folate metabolism in rat pancreas: nitrous oxide inactivation of methionine synthase leads to accumulation of 5-methyltetrahydrofolate in cytosol.

Folate-dependent one-carbon metabolism and methylation reactions have been implicated in the secretory function of the pancreas. Because vitamin B-12 deficiency perturbs folate metabolism, we determined the effects of nitrous oxide inactivation of methionine synthase on the compartmentation of folate metabolism in rat pancreas. Rats were exposed to an atmosphere of nitrous oxide and oxygen (80 and 20%, respectively) for 18 h; control rats breathed air. Folate coenzyme concentrations were determined by HPLC and Lactobacillus casei microbiological assay of the cytosolic and mitochondrial fractions of pancreas, which contained 62 and 46%, respectively, of the total folate. In pancreas of control rats, cytosolic folates were 5-methyltetrahydrofolate (31% of total folates), tetrahydrofolate (54%) and 5- and 10-formyltetrahydrofolate (6 and 8%, respectively). In the rats exposed to nitrous oxide, cytosolic 5-methyltetrahydrofolate concentrations were significantly greater (59% of total folates) and tetrahydrofolate concentrations were significantly lower (32%) than in controls; however, total cytosolic folate levels were unaffected by nitrous oxide exposure. In controls, mitochondrial folates were composed of 5-methyltetrahydrofolate (9% of total folates), tetrahydrofolate (60%) and 5- and 10-formyltetrahydrofolate (22 and 10%, respectively). Exposure to nitrous oxide led to significantly lower total mitochondrial folates (1.49 +/- 0.18 vs. 0.75 +/- 0.29 nmol/g, control vs. nitrous oxide, P < 0.05). This was due to a significantly lower concentration of tetrahydrofolate and 5-formyltetrahydrofolate, but not of 5-methyl- or 10-formyltetrahydrofolate. The activity of methionine synthase was 85% lower (P < 0.001) in pancreatic extracts of rats exposed to nitrous oxide than in controls. These results show that cytosolic folates accumulate in pancreas as the 5-methyl derivative at the expense of other reduced folates, as happens in liver. However, in contrast to results in liver, the mitochondrial folate concentration was lower in the pancreas of rats exposed to nitrous oxide, and this decline was limited to the 5-formyl- and tetrahydrofolate derivatives.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1989

RNA metabolism in nuclei: adenovirus and heat shock alter intranuclear RNA compartmentalization.

Nuclear RNA compartmentalization has received little attention as an area where regulation of gene expression could occur. RNA transcription and processing occur in association with the nuclear matrix, a salt-insoluble proteinaceous network that fills the nuclear space and is contiguous with the peripheral lamina and pore complexes. Described here are experiments that determine the fate of nuclear RNA after it has completed these matrix-associated maturation steps. Continuous label experiments indicate that after nuclear RNA is processed it changes its state of attachment in the nucleus so that it is now removed from the nucleus in the high salt extraction step of matrix isolation. It is this salt-extractable RNA that will be transported to the cytoplasm. Late in adenovirus infection and following heat shock, when transport of cellular RNA is decreased, cellular transcripts do not make the transition from the matrix-associated to the salt-extractable nuclear pool. The implication of these data for the regulation of gene expression is discussed.

Adenoviruses, Human↗
PubMed1992

Metabolic compartmentation of vertebrate glutamine synthetase: putative mitochondrial targeting signal in avian liver glutamine synthetase.

The evolution of uricoteley as a mechanism for hepatic ammonia detoxication in vertebrates required targeting of glutamine synthetase (GS) to liver mitochondria in the sauropsid line of descent leading to the squamate reptiles and archosaurs. Previous studies have shown that in birds and crocodilians, sole survivors of the archosaurian line, hepatic GS is translated without a transient, N-terminal targeting signal common to other mitochondrial matrix proteins. To identify a putative internal targeting sequence in the avian enzyme, the amino acid sequence of chicken liver GS was derived by a combination of sequencing of cloned cDNA, direct sequencing of mRNA, and sequencing of polymerase chain reaction (PCR) products amplified from reverse-transcribed mRNA. Analysis of the first 20 or so N-terminal amino acids of the derived sequence for the chicken enzyme shows that they are devoid of acidic amino acids, contain several hydroxy amino acids, and can be predicted to form a positively charged, amphipathic helix, all of which are characteristic properties of mitochondrial targeting signals. A comparison of the N-terminus of chicken GS with the N-termini of cytosolic mammalian GSs indicates that at least three amino acid replacements may have been responsible for converting the N-terminus of the cytosolic mammalian enzyme into a mitochondrial targeting signal. Two of these, His15 and Lys19, result in additional positive charges, as well as in changes in hydrophilicity. Both could have resulted from third-base-codon substitutions. A third replacement, Ala12, may contribute to the helicity of the N-terminus of the chicken enzyme. The N-terminus of the cytosolic chicken brain GS (positions 1-36) was found to be identical to that of the liver enzyme. The complete sequence of chicken retinal GS is also identical to that of the liver enzyme. GS is coded by a single gene in birds, so these sequence data suggest that, unlike the situation in other tissue-specific compartmental isozymes, differential targeting of avian GS to the mitochondrial or cytosolic compartments is not dependent on the sequence of the primary translation product of its mRNA but may involve some other tissue-specific factor(s).

Amino Acid Sequence↗
PubMed2000

Purification, characterization, and biological compartmentalization of rat fetal antigen 1.

This study has established the rat as an animal model for the analysis of the biological role of fetal antigen 1 (FA1), a protein previously described in humans and mice. FA1 was purified from rat amniotic fluid by immunospecific affinity chromatography. Immunochemical identity between mouse and rat FA1 was established by crossed tandem immunoelectrophoresis. Molecular size was analyzed by mass spectrometry (33 kDa). The amino acid composition was determined, and the amino acid sequence was analyzed. The overall amino acid composition and sequence of the 28 first N-terminal amino acids were identical to the corresponding parts of rat preadipocyte factor 1 and rat adrenal zona glomerulosa protein. Extensive sequence similarity was found between rat and mouse FA1 (86%) and between rat and human FA1 (82%). The concentration of FA1 in fetal serum, maternal serum, urine, and amniotic fluid in rats was determined using an ELISA. The highest concentrations were found in fetal serum and amniotic fluid around Day 18 of pregnancy. This is the first report on the physicochemical characteristics and compartmentalization of rat FA1.

Amino Acid Sequence↗
PubMed2002

Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells.

Fluorescence microscopy of A549 cells stained with a glutathione (L-gamma-glutamyl-L-cysteinylglycine, GSH)-specific polyclonal antibody displayed uniform staining of the peri-nuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol-reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)-dependent inhibition of GSH synthesis. However, confocal visualization of antibody-stained A549 cells in the z-plane revealed the majority of the peri-nuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z-plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z-section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near-complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z-plane. The antibody also provided initial visualization of the compartmentalization of protein-GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti-GSH antibody-stained Jurkat T lymphocytes, we also demonstrated population variations in the cellular compliment of GSH and protein-GSH mixed disulfides, formed in response to diamide. In addition, we showed cell-cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.

Animals↗
PubMed2003

Cyclic AMP compartmentation due to increased cAMP-phosphodiesterase activity in transgenic mice with a cardiac-directed expression of the human adenylyl cyclase type 8 (AC8).

Hearts from AC8TG mice develop a higher contractility (LVSP) and larger Ca2+ transients than NTG mice, with (surprisingly) no modification in L-type Ca2+ channel current (ICa,L) (1). In this study, we examined the cardiac response of AC8TG mice to beta-adrenergic and muscarinic agonists and IBMX, a cyclic nucleotide phosphodiesterase (PDE) inhibitor. Stimulation of LVSP and ICa,L by isoprenaline (ISO, 100 nM) was twofold smaller in AC8TG vs. NTG mice. In contrast, IBMX (100 microM) produced a twofold higher stimulation of ICa,L in AC8TG vs. NTG mice. IBMX (10 microM) increased LVSP by 40% in both types of mice, but contraction and relaxation were hastened in AC8TG mice only. Carbachol (10 microM) had no effect on basal contractility in NTG hearts but decreased LVSP by 50% in AC8TG mice. PDE assays demonstrated an increase in cAMP-PDE activity in AC8TG hearts, mainly due to an increase in the hydrolytic activity of PDE4 and PDE1 toward cAMP and a decrease in the activity of PDE1 and PDE2 toward cGMP. We conclude that cardiac expression of AC8 is accompanied by a rearrangement of PDE isoforms, leading to a strong compartmentation of the cAMP signal that shields L-type Ca2+ channels and protects the cardiomyocytes from Ca2+ overload.

1-Methyl-3-isobutylxanthine↗