Anti-inflammatory effects of macrolide antibiotics.

Macrolides are widely used as antibacterial drugs. Clinical and experimental data, however, indicate that they also modulate inflammatory responses, both contributing to the treatment of infective diseases and opening new opportunities for the therapy of other inflammatory conditions. Considerable evidence, mainly from in vitro studies, suggests that leukocytes and neutrophils in particular, are important targets for modulatory effects of macrolides on host defense responses. This underlies the use of the 14-membered macrolide erythromycin for the therapy of diffuse panbronchiolitis. A variety of other inflammatory mediators and processes are also modulated by macrolides, suggesting that the therapeutic indications for these drugs may be extended significantly in future.

Metabolic adaptation of endothelial cells to substrate deprivation.

Endothelial cells are known to be metabolically rather robust. To study the mechanisms involved, porcine aortic endothelial cells (PAEC), cultured on microcarrier beads, were perfused with glucose (10 mM) or with substrate-free medium. Substrate-free perfusion for 2 h induced an almost complete loss of nucleoside triphosphates (31P-NMR) and decreased heat flux, a measure of total energy turnover, by >90% in parallel microcalorimetric measurements. Heat flux and nucleoside triphosphates recovered after addition of glucose. Because protein synthesis is a major energy consumer in PAEC, the rate of protein synthesis was measured ([14C]leucine incorporation). Reduction or blockade of energy supply resulted in a pronounced reduction in the rate of protein synthesis (up to 80% reduction). Intracellular triglyceride stores were decreased by approximately 60% after 2 h of substrate-free perfusion. Under basal perfusion conditions, PAEC released approximately 30 pmol purine. mg protein-1. min-1, i.e., 16% of the cellular ATP per hour, while ATP remained constant. Substrate deprivation increased the release of various purines and pyrimidines about threefold and also induced a twofold rise in purine de novo synthesis ([14C]formate). These results demonstrate that PAEC are capable of recovering from extended periods of substrate deprivation. They can do so by a massive downregulation of their energy expenditure, particularly protein synthesis, while at the same time using endogenous triglycerides as substrates and upregulating purine de novo synthesis to compensate for the loss of purines.

Purinogen is not an endogenous substrate used in endothelial cells during substrate deprivation.

Porcine aortic endothelial cells (PAEC) are known to be metabolically robust. They are capable of surviving extended periods of complete lack of exogenous substrate, and purine release has been shown to be significantly up-regulated. The endogenous substrates used during substrate deprivation, as well as the sources responsible for the increased purine release, have not been completely identified. We tested the possibility that a phosphoglyceroyl-ATP-containing polymer, purinogen, might support PAEC hibernation induced by lack of exogenous substrate. This involved isolation of the acid-insoluble fraction of PAEC, which was presumed to contain purinogen, and analysis by HPLC and 31P NMR. No evidence supporting the presence of triphosphate-containing compounds (purinogen) was found. Similar results were obtained in the rat heart. The majority of the products in the acid-insoluble, alkaline-treated fraction were identified as RNA degradation products (2'- and 3'-nucleoside monophosphates). A [14C]adenosine labelling experiment showed that incorporation of adenosine into the acid-insoluble fraction was almost completely prevented after inhibition of RNA synthesis with actinomycin D. Furthermore, RNA isolated from PAEC and subsequently treated with alkali showed a profile that was almost identical with the HPLC profile of the acid-insoluble fraction. Finally, substrate-free incubation of the cells did not quantitatively or qualitatively influence the distribution of acid-insoluble derivatives. We conclude that PAEC survival during the absence of exogenous substrate is not supported by purinogen but rather by some other, yet-to-be-identified, endogenous substrate.

Regulation of endothelial Na(+)-K(+)-ATPase activity by cAMP.

Using an in vitro cell system and Cs+ NMR techniques we were able to show that porcine aortic endothelial cells (PAEC) reduce their Na(+)-K(+)-ATPase activity upon an increase in intracellular cAMP. Reduction in the pump rate was due to phosphorylation of the alpha-subunit of the ATPase as shown by immunoprecipitation. Apart from a pump inhibiton using 8-Br-cAMP and IBMX, we were also able to show that changes in the Na(+)-K(+)-ATPase activity could be mediated by the adenosine-A2 and prostaglandin receptor agonists 5'-N-Ethylcarboxamidoadenosine and Iloprost, respectively. Parallel to a decrease in pump activity we also observed a decrease in intracellular Cs+, indicating opening of K+ channels.

Energy turnover of vascular endothelial cells.

Two noninvasive methods, calorimetry and 31P nuclear magnetic resonance (NMR), were used to further define energy-consuming and energy-providing reactions in endothelial cells. With 31P-NMR, cellular ATP content was measured; with calorimetry, heat flux as a result of ATP turnover was measured. For these measurements, pig aortic endothelial cells were cultured on microcarrier beads and perfused in a column at constant flow rate. Pig aortic endothelial cells synthesize ATP mainly through glycolysis and, as determined by NMR, contain no phosphocreatine. In such a system, calorimetry-measured heat flux reflects rate of cellular ATP turnover. By use of inhibitors of ATP-dependent processes, the following changes in basal heat flux (231 +/- 65.5 microW/mg protein) were obtained: 18% for 2,3-butanedione monoxime (inhibitor of actomyosin-ATPase), 17% for wortmannin (inhibitor of myosin light chain kinase), 10% for cytochalasin D (inhibitor of actin polymerization), 23% for cycloheximide (inhibitor of protein synthesis), 11% for thapsigargin (inhibitor of endoplasmic reticulum Ca(2+)-ATPase), and 6% for bafilomycin A1 (inhibitor of lysosomal H(+)-ATPase). Cytochalasin D, 2,3-butanedione monoxime, wortmannin, and thapsigargin caused changes in F-actin distribution, as revealed by rhodamine-phalloidin cytochemistry. In a separate experimental series, when cells were perfused with a medium containing no glucose, heat flux decreased by 40% while cellular ATP remained unchanged. Inhibition of glycolysis with 2-deoxy-D-glucose decreased heat flux by 73%, and ATP was no longer visible with 31P-NMR. Despite this massive ATP depletion, which was maintained for 3 h, cells fully recovered heat flux and ATP when 2-deoxy-D-glucose was removed. The results, together with previously published data for Na(+)-K(+)-ATPase [M. L. H. Gruwel, C. Alves, and J. Schrader. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H351-H358, 1995], demonstrate that > 70% of total ATP-consuming processes of endothelial cells can be attributed to specific cellular processes. Actomyosin-ATPase (18%) and protein synthesis (23%) comprise the largest fraction. At least three-fourths of ATP synthesized is provided by glycolysis. Endothelial cells exhibit the remarkable ability to coordinate downregulation of ATP synthesis and consumption when glycolysis is inhibited.

A 133Cs nuclear magnetic resonance study of endothelial Na(+)-K(+)-ATPase activity: can actin regulate its activity?

Using (133)Cs+ NMR, we developed a technique to repetitively measure, in vivo, Na(+)-K(+)-ATPase activity in endothelial cells. The measurements were made without the use of an exogenous shift reagent, because of the large chemical shift of 1.36 +/- 0.13 ppm between intra- and extracellular Cs+. Intracellularly we obtained a spin lattice relaxation time (T1) of 2.0 +/- 0.3 s, and extracellular T1 was 7.9 +/- 0.4 s. Na(+)-K+ pump activity in endothelial cells was determined at 12 +/- 3 nmol Cs+ x min(-1) x (mg Prot)[-1] under control conditions. When intracellular ATP was depleted by the addition of 5 mM 2-deoxy-D-glucose (DOG) and NaCN to about 5% of control, the pump rate decreased by 33%. After 80 min of perfusion with 5 mM DOG and NaCN, reperfusion with control medium rapidly reestablished the endothelial membrane Cs+ gradient. Using (133)Cs+ NMR as a convenient tool, we further addressed the proposed role of actin as a regulator of Na(+)-K+ pump activity in intact cells. Two models of actin rearrangement were tested. DOG caused a rearrangement of F-actin and an increase in G-actin, with a simultaneous decrease in ATP concentration. Cytochalasin D, however, caused an F-actin rearrangement different from that observed for DOG and an increase in G-actin, and cellular ATP levels remained unchanged. In both models, the Na(+)-K(+)-pump activity remained unchanged, as measured with (133)Cs NMR. Our results demonstrate that (133)Cs NMR can be used to repetitively measure Na(+)-K(+)-ATPase activity in endothelial cells. No evidence for a regulatory role of actin on Na(+)-K(+)-ATPase was found.

Regulation of proliferation of LLC-MK2 cells by nucleosides and nucleotides: the role of ecto-enzymes.

1. Using the incorporation of [methyl-3H]thymidine as a proliferation marker, the effects of various nucleosides and nucleotides on endothelial LLC-MK2 cells were studied. We found that ATP, ADP, AMP and adenosine in concentrations of 10 microM or higher stimulate the proliferation of these cells. 2. Inhibition of ecto-ATPase (EC 3.6.1.15), 5'-nucleotidase (EC 3.1.3.5) or alkaline phosphatase (EC 3.1.3.1) significantly diminished the stimulatory effect of ATP, indicating that the effect is primarily caused by adenosine and not by adenine nucleotides. Also, the effect depends only on extracellular nucleosides, since inhibition of nucleoside uptake by dipyridamole has no influence on proliferation. 3. Other purine nucleotides and nucleosides (ITP, GTP, inosine and guanosine) also stimulate cell proliferation, while pyrimidine nucleotides and nucleosides (CTP, UTP, cytidine and uridine) inhibit proliferation. Furthermore, the simultaneous presence of adenosine and any of the other purine nucleosides is not entirely additive in its effect on cell proliferation. At the same time any pyrimidine nucleoside, when added together with adenosine, has the same inhibitory effect as the pyrimidine nucleoside alone. 4. Apparently these proliferative effects are neither caused by any pharmacologically known P1-purinoceptor, nor are they mediated by cyclic AMP, cyclic GMP, or D-myo-inositol 1,4,5-trisphosphate as second messenger, nor by extracellular Ca2+. 5. Therefore, we conclude that various purine and pyrimidine nucleosides can influence the proliferation of LLC-MK2 cells by acting on putative purinergic and pyrimidinergic receptors not previously described.

Autoradiography-based cytochemical detection of ecto-ATPase, ecto-ADPase, 5'-nucleotidase, and extracellular adenosine production, employing 141Ce3+ as a capturing agent.

A method for the visualization of the ecto-nucleotidase enzyme activities present on the cell surface, employing 141Ce3+ as a capturing and labelling agent, is described. Phosphate ions precipitated at the cell surface can be detected by coating the cells with an autoradiographic emulsion, followed by light microscopical inspection of the formed silver grains. The activities of ecto-ATPase, ecto-ADPase and 5'-nucleotidase were detected by this approach in four different cell lines. Parallel biochemical measurements of the activities of the corresponding enzymes were carried out in order to validate, evaluate, and optimize the cytochemical detection. The finding that Ce3+ ions are inhibitory to ecto-ATPase provided evidence for the necessity of carefully establishing appropriate reaction conditions for the cytochemical determination of ecto-nucleotidases. The application of this method to the indirect detection of extracellular adenosine production from substrates like ATP has also been documented. It allows a cytochemical determination of adenosine formed through cascade nucleotide dephosphorylation. This newly described method is of high sensitivity and potentially of value for a variety of applications, including not only cytochemistry but also cell biology, and molecular biology studies.

Sperm motility and kinetics of dynein ATPase in astheno- and normozoospermic samples after stimulation with adenosine and its analogues.

We tested the effects of adenosine and 2-deoxyadenosine on the activation of human spermatozoa. In the asthenozoospermic group of patients adenosine produces an increase in sperm motility from 33.3 +/- 2.1% to 42.1 +/- 3.4%, progressive motility from 22.5 +/- 1.3% to 28.6 +/- 1.7% and forward progression rating from 2.1 +/- 0.2% to 2.8 +/- 0.1%. 2-Deoxyadenosine stimulated asthenozoospermic samples to a greater degree than adenosine. Sperm motility rose to 48.9 +/- 3.4%, progressive motility to 32.1 +/- 3.4% and forward progression rating to 3.0 +/- 0.1% following stimulation with 2-deoxy-adenosine. The kinetic parameters and basic characteristics of dynein ATPase were determined. The maximum activity of dynein ATPase, Vmax, was significantly different (P < 0.001) for asthenozoospermic and normozoospermic samples: 6.46 +/- 2.1 nmol Pi/mg/min and 16.99 +/- 3.7 nmol Pi/mg/min respectively. However, the enzyme affinity for ATP was not different. Stimulation of asthenozoospermic samples with adenosine and 2-deoxyadenosine caused an increase of Vmax (70-90% and 90-110% respectively) and no significant change in KM was observed. In order to block the nucleoside transporter and to eliminate the action of adenosine inside the cell, dipyridamole was used but the effects of adenosine were not neutralized. 5'-(N-ethylcarboxy-amido)-adenosine showed effects similar to those of adenosine, even when applied in 1 microM concentration. These results indicate that adenosine and its analogues stimulate sperm motility and activity of dynein ATPase, most probably via A2 receptors.

The cytoplasmic domain of C-CAM is required for C-CAM-mediated adhesion function: studies of a C-CAM transcript containing an unspliced intron.

Cell-CAM105 (also named C-CAM) is a cell surface glycoprotein involved in intercellular adhesion of rat hepatocytes. It has four extracellular immunoglobulin (Ig) domains, a transmembrane domain and a cytoplasmic domain and therefore is a member of the Ig supergene family. We have characterized multiple cDNAs of the C-CAM genes in rat intestine. Sequence analyses showed that rat intestine contained not only the previously reported L-form and S-form C-CAMs (renamed C-CAM1 and C-CAM2 respectively) but also a new isoform, C-CAM3. The C-CAM3 transcript codes for a polypeptide with a truncated C-terminus that lacks 65 amino acids from the previously reported C-CAM1 cytoplasmic domain. Unlike C-CAM1, C-CAM3 did not mediate cell adhesion when expressed in insect cells using the baculoviral expression system. Thus the extra 65 amino acids in the cytoplasmic domain of C-CAM1 are important for adhesion phenotype when expressed in insect cells. Although C-CAM1 and C-CAM2 are encoded by different genes, sequence analysis suggests that C-CAM3 is probably derived from alternative splicing of the C-CAM1 gene. To examine this possibility, we have determined the exon organization of the C-CAM1 gene. C-CAM3 differed from C-CAM1 by the presence of a single unspliced intron which contained a stop codon immediately after the regular splice junction. As a result, translation of C-CAM3 terminates at the point where C-CAM1 and C-CAM3 sequences diverge. To investigate the expression of C-CAM1, C-CAM2 and C-CAM3 in different tissues, we used an RNAase-protection assay to simultaneously assess the levels of expression of these transcripts. Using total RNA prepared from various tissues, we showed that expression of C-CAM3 was tissue-specific, and the C-CAM3 transcript accounted for about 25% of the transcripts derived from the C-CAM1 gene. However, further analysis revealed that C-CAM3 transcript was not present in cytosolic RNA, rather it was enriched in nuclear RNA prepared from hepatocytes. Although C-CAM3 cDNA contains the polyadenylation signal and is polyadenylated, these results indicate that C-CAM3 is probably an incomplete spliced product of C-CAM1 gene.

Cell-CAM105 isoforms with different adhesion functions are coexpressed in adult rat tissues and during liver development.

The rat hepatocyte cell adhesion molecule cell-CAM105 has recently been shown to be composed of at least two isoforms. Expression of the two isoforms in different tissues and during fetal liver development in rats was studied by RNase protection using a probe which could specifically and simultaneously detect both isoforms. This probe revealed protected fragments of expected lengths for the L-form and the S-form in RNA samples isolated from various adult rat tissues. High levels of the L-form and S-form messages were detected in liver and intestine, moderate levels were detected in lung, and weak signals were detected in muscle, kidney, and spleen. In liver development studies, the messages for cell-CAM105 showed a major increase on the first day after birth compared to the fetal stage, and both isoform messages were proportionally increased. These results indicate that both cell-CAM105 isoforms may have function(s) related to hepatocyte differentiation. To study the adhesion function of cell-CAM105 isoforms, full-length cDNAs for these isoforms were expressed in insect cells. The insect cells expressing the L-form cell-CAM105 were found to aggregate. However, expression of S-form cell-CAM105 did not support cell aggregation. These results indicate that L-form, but not S-form, cell-CAM105 directly mediates the cell adhesion function.

Molecular cloning and expression of a new rat liver cell-CAM105 isoform. Differential phosphorylation of isoforms.

An hepatocyte cell-adhesion molecule (cell-CAM105) was recently shown to be identical with the liver plasma-membrane ecto-ATPase. This protein has structural features of the immunoglobulin superfamily and is homologous with carcinoembryonic antigen proteins. We have cloned a cDNA encoding a new form of the cell-CAM105 which is a variant of the previously isolated clone. In addition to having a shorter cytoplasmic domain, the new isoform also has substitutions clustered in the first 130 amino acids of the extracellular domain. Both of these isoforms are expressed on the surface of hepatocytes with the shorter variant being the predominant form. The previously isolated cell-CAM105 (long form) has more potential phosphorylation sites than does the new isoform (short form). Both isoforms are found to be phosphorylated after incubation with [32P]phosphate in vitro, with the long form being phosphorylated to a significantly higher extent. This observed differential phosphorylation could be one of the mechanisms for the regulation of isoform functions. Using antipeptide antibodies specific for the long form and antibodies that are reactive with both isoforms, we have shown that both isoforms are localized in the canalicular domain of hepatocytes. The sequence differences between these two isoforms suggest that they are probably derived from different genes rather than from alternative splicing.

Localization of ecto-ATPase in rat kidney and isolated renal cortical membrane vesicles.

Brush-border (BBMV) and basolateral membrane vesicles (BLMV) from rat renal cortex exhibit an ecto-ATPase activity that is distinct from other ATPases. We have examined the cellular and regional distribution of this enzyme in rat kidney using antibodies against rat liver ecto-ATPase. In isolated vesicles, the distribution shown by biochemical assays of ATPase activity was confirmed by immunocytochemistry and Western blotting. Indirect immunofluorescence and immunogold labeling showed that brush borders of the S1 and S3 segments of the proximal tubule (PT) were stained, but the S2 segment was negative. Staining was most intense in the S3 segment. The luminal membrane of the initial part of the thin descending limb of Henle also showed a marked staining. Surprisingly, basolateral plasma membranes of PT had no detectable staining. However, the plasma membrane of endothelial cells was heavily stained, both in larger vessels and in peritubular capillaries. Using an antibody against rat thrombomodulin, a marker for endothelial cell plasma membranes, we showed that preparations of BBMV, BLMV, and endocytic vesicles are all contaminated with these membranes. This may explain, at least partially, the biochemically measured ecto-ATPase activity in renal cortical membrane vesicles. Finally, no specific staining in the kidney was found using polyclonal antipeptide antibodies against the "long form" of liver ecto-ATPase, either by immunocytochemistry or by Western blotting. This indicates either that there is no long isoform of the ecto-ATPase in the kidney or that the intracellular domains of the long form are different in the two tissues.

Immunochemical characterization of two isoforms of rat liver ecto-ATPase that show an immunological and structural identity with a glycoprotein cell-adhesion molecule with Mr 105,000.

One of the cell-adhesion molecules (CAMs) responsible for rat hepatocyte aggregation has been described as a glycoprotein having an Mr of 105,000 (cell-CAM105). The Mr and localization of cell-CAM105 in liver membranes are very similar to those of liver ecto-ATPase, an ATPase with its nucleotide-hydrolysing site localized on the outside of the cell membrane. The protein sequence of the ecto-ATPase has been deduced from cDNA cloning. Structural analysis of the sequence indicates that the ecto-ATPase has immunoglobulin-like domains and is a member of the immunoglobulin superfamily. Since a group of proteins in the immunoglobulin superfamily has been shown to have functions related to cell adhesion, the structural characteristics of the ecto-ATPase further led to the possibility that the ecto-ATPase may have functions related to cell adhesion. In this paper, using the cDNA for the ecto-ATPase, the anti-peptide antibodies produced against peptides derived from the ecto-ATPase cDNA sequence and monoclonal antibodies against the cell-CAM105, we present evidence of identity between cell-CAM105 and ecto-ATPase. First, in Western immunoblots, two anti-cell-CAM105 monoclonal antibodies cross-reacted with the purified ecto-ATPase. Secondly, in immunodepletion experiments, antibodies against the ecto-ATPase depleted the same protein recognized by the anti-cell-CAM105 antibodies. Thirdly, in two-dimensional gel-electrophoretic analysis, anti-peptide antibodies generated against an extracellular N-terminal peptide and the intracellular C-terminal peptides of the ecto-ATPase immunoprecipitated proteins of similar isoelectric points and Mr values to those of the cell-CAM105. Fourthly, proteins immunoprecipitated by anti-ecto-ATPase antibodies and anti-cell-CAM105 antibodies have similar V8-proteinase-digest peptide maps. Finally, monoclonal antibodies against the cell-CAM105 specifically recognized the protein expressed in COS cells transfected with the ecto-ATPase cDNA. These results indicate that the ecto-ATPase cDNA codes for a protein that is identical with the cell-CAM105. Since the ecto-ATPase has structural features of immunoglobulin domains, the identity of cell-CAM105 with ecto-ATPase leads to the conclusion that this liver CAM, similarly to neuronal CAM, is also a member of the immunoglobulin supergene family. Furthermore, immunological studies indicate that the cell-CAM105/ecto-ATPase is composed of two isoforms of different C-terminal sequences. The association of ATPase activity with cell-CAM105 raises the possibility that extracellular nucleotides may play important roles in regulating cell adhesion.

The stepwise hydrolysis of adenine nucleotides by ectoenzymes of rat renal brush-border membranes.

Evidence is presented for the existence of ectoenzymes in rat renal cortical brush-border membrane vesicles that produce adenosine as a final product using either ATP, ADP or AMP as substrate. The enzymes are insensitive to levamisole, ouabain, oligomycin and N-ethylmaleimide, and have absolute requirement for divalent cations with following order of activation Mg2+ greater than Ca2+ greater than Mn2+ greater than Ba2+ greater than Zn2+. At least two separate enzymes can be distinguished. One is capable of hydrolyzing ATP, other nucleoside triphosphates and ADP, but not AMP. The enzyme is insensitive to concanavalin A. The other enzyme hydrolyzes AMP and is strongly inhibited by this lectin. Mg2(+)-stimulated ATP hydrolysis displays saturation kinetics which is not of the simple Michaelis-Menten type, but is biphasic with a high-affinity (K'm = 0.16 mM) and low-affinity site (K'm = 9.0 mM), respectively. The low-affinity site hydrolyzes ATP, ITP and GTP to a similar extent, whereas CTP and UTP with about 40% lower rate. The high-affinity site splits ATP much better than other nucleoside triphosphates. Hydrolysis of ADP follows simple Michaelis-Menten saturation kinetic with apparent Km = 0.38 +/- 0.06 mM. Inhibition, activation and substrate specificity studies indicate that nucleoside triphosphatase and nucleoside diphosphatase may reside on the same protein. Kinetics of the AMP hydrolysis is hyperbolic with apparent Km = 76 +/- 9 microM. The cascade of ectonucleotidases in the brush-border membrane of the proximal tubule may catalyze the degradation of filtered nucleotides into adenosine and phosphate, the compounds which are thereafter probably reabsorbed by separate transport systems.