Enhancement of collagen deposition and cross-linking by coupling lysyl oxidase with bone morphogenetic protein-1 and its application in tissue engineering.

Cultured cell-derived extracellular matrices (ECM)-based biomaterials exploit the inherent capacity of cells to create highly sophisticated supramolecular assemblies. However, standard cell culture conditions are far from ideal given the fact that the diluted microenvironment does not favor the production of ECM components, a circumstance particularly relevant for collagen. An incomplete conversion of procollagen by C-proteinase/bone morphogenetic protein 1 (BMP1) has been proposed to severely limit in vitro collagen deposition. BMP1 also catalyzes the proteolytic activation of the precursor of the collagen cross-linking enzyme, lysyl oxidase (LOX) to yield the active form, suggesting a deficit in cross-linking activity under standard conditions. We hypothesized that the implementation of fibroblast cultures with LOX and BMP1 may be an effective way to increase collagen deposition. To test it, we have generated stable cell lines overexpressing LOX and BMP1 and studied the effect of supernatants enriched in LOX and BMP1 on collagen synthesis and deposition from fibroblasts. Herein, we demonstrate that the supplementation with LOX and BMP1 strongly increased the deposition of collagen onto the insoluble matrix at the expense of the soluble fraction in the extracellular medium. Using decellularization protocols, we also show that fibroblast-derived matrices regulate adipogenic and osteogenic differentiation of human mesenchymal stem cells (MSC), and this effect was modulated by LOX/BMP1. Collectively, these data demonstrate that we have developed a convenient protocol to enhance the capacity of in vitro cell cultures to deposit collagen in the ECM, representing this approach a promising technology for application in tissue engineering.

Elevated expression levels of lysyl oxidases protect against aortic aneurysm progression in Marfan syndrome.

Patients with Marfan syndrome (MFS) are at high risk of life-threatening aortic dissections. The condition is caused by mutations in the gene encoding fibrillin-1, an essential component in the formation of elastic fibers. While experimental findings in animal models of the disease have shown the involvement of transforming growth factor-ß (TGF-ß)- and angiotensin II-dependent pathways, alterations in the vascular extracellular matrix (ECM) may also play a role in the onset and progression of the aortic disease. Lysyl oxidases (LOX) are extracellular enzymes, which initiates the formation of covalent cross-linking of collagens and elastin, thereby contributing to the maturation of the ECM. Here we have explored the role of LOX in the formation of aortic aneurysms in MFS. We show that aortic tissue from MFS patients and MFS mouse model (Fbn1(C1039G/+)) displayed enhanced expression of the members of the LOX family, LOX and LOX-like 1 (LOXL1), and this is associated with the formation of mature collagen fibers. Administration of a LOX inhibitor for 8weeks blocked collagen accumulation and aggravated elastic fiber impairment, and these effects correlated with the induction of a strong and rapidly progressing aortic dilatation, and with premature death in the more severe MFS mouse model, Fbn1(mgR/mgR), without any significant effect on wild type animals. This detrimental effect occurred preferentially in the ascending portion of the aorta, with little or no involvement of the aortic root, and was associated to an overactivation of both canonical and non-canonical TGF-ß signaling pathways. The blockade of angiotensin II type I receptor with losartan restored TGF-ß signaling activation, normalized elastic fiber impairment and prevented the aortic dilatation induced by LOX inhibition in Fbn1(C1039G/+) mice. Our data indicate that LOX enzymes and LOX-mediated collagen accumulation play a critical protective role in aneurysm formation in MFS.

Papel del eje TGF-beta-endotelina 1 en la fibrosis: lecciones del endotelio y el fibroblasto / No disponible

No disponible

Screening of the endothelin1 gene (EDN1) in a cohort of patients with essential left ventricular hypertrophy.

Our objective was to analyse the role of endothelin1 gene (EDN1) variation in essential left ventricular hypertrophy (LVH). We searched for EDN1 variants in 145 Spanish patients with an essential form of LVH (not secondary to hypertension, aortic stenosis, or any other disease that could explain the hypertrophy). The five EDN1 coding exons and 1.5 kilobases of the promoter region were analysed through single strand conformation analysis and direct sequencing. We found four nucleotide changes: -1224 C/A (promoter), -131 ins/del A (exon 1, 5'-non-translated sequence), A/G in codon 106 (exon 3, silent), and G/T in codon 198 (exon 5, lys198asn). To determine the association between these polymorphisms and cardiac hypertrophy, we compared the genotype frequencies from these 145 patients with 250 healthy controls. We found a higher frequency of patients homozygous for 198 lys (198 KK) (65% vs. 52%; p = 0.01; OR = 1.76) and for -1224 AA (73% vs. 66%; p = 0.19). Homozygotes for -1224 A + 198 K (AA+KK) were significantly more frequent in patients (62% vs. 45%; p = 0.0007; OR = 2.10; 95% CI = 1.35-3.25). The expression of the -1224 C/A and exon 5 K198N variants was analysed with cells in culture. These in vitro studies showed that these variations did not differ in their expression levels. In conclusion, our work has shown that EDN1 variation, and in particular homozygosity for the -1224A/198K haplotype, is associated with the risk of developing cardiac hypertrophy. However, these EDN1 variants do not affect in vitro gene expression.

Posttranscriptional regulation of human iNOS by the NO/cGMP pathway.

Nitric oxide (NO) and cGMP may exert positive or negative effects on inducible NO synthase (iNOS) expression. We have explored the influence of the NO/cGMP pathway on iNOS levels in human mesangial cells. Inhibition of NOS activity during an 8-h stimulation with IL-1beta plus tumor necrosis factor (TNF)-alpha reduced iNOS levels, while NO donors amplified iNOS induction threefold. However, time-course studies revealed a subsequent inhibitory effect of NO donors on iNOS protein and mRNA levels. This suggests that NO may contribute both to iNOS induction and downregulation. Soluble guanylyl cyclase (sGC) activation may be involved in these effects. Inhibition of sGC attenuated IL-1beta/TNF-alpha-elicited iNOS induction and reduced NO-driven amplification. Interestingly, cGMP analogs also modulated iNOS protein and mRNA levels in a biphasic manner. Inhibition of transcription unveiled a negative posttranscriptional modulation of the iNOS transcript by NO and cGMP at late times of induction. Supplementation with 8-bromo-cGMP (8-BrcGMP) reduced iNOS mRNA stability by 50%. These observations evidence a complex feedback regulation of iNOS expression, in which posttranscriptional mechanisms may play an important role.

Nitric oxide-sensitive guanylyl cyclase activity inhibition through cyclic GMP-dependent dephosphorylation.

The soluble form of guanylyl cyclase (sGC) plays a pivotal role in the transduction of inter- and intracellular signals conveyed by nitric oxide. Here, a feedback inhibitory mechanism triggered by cyclic guanosine-3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) activation is described. Preincubation of chromaffin cells with C-type natriuretic peptide, which increased cGMP levels and activated PKG, or with cGMP-permeant analogue (which also activates PKG), in the presence of a broad-spectrum phosphodiesterase inhibitor, resulted in a decrease in subsequent sodium nitroprusside (SNP)-dependent cGMP elevations. This inhibitory effect was mimicked by activating a protein phosphatase and counteracted by the selective PKG inhibitor KT-5823 and by different protein phosphatase inhibitors. Immunoprecipitation of sGC from cells submitted to different treatments followed by immunodetection with antiphosphoserine antibodies (clone 4A9) showed changes in phosphorylation levels of the beta subunit of sGC, and these changes correlated well with differences in SNP-elicited cGMP accumulations. Pretreatment of cells with several PKG inhibitors or protein phosphatase inhibitors produced an enhancement of SNP-stimulated cGMP rises without changing the SNP concentration required to produce half-maximal or maximal responses. Taken together, these results indicate that the catalytic activity of sGC is closely coupled to the phosphorylation state of its beta subunit and that the tonic activity of PKG or its stimulation regulates sGC activity through dephosphorylation of the beta subunit.

Inhibition of small G proteins of the rho family by statins or clostridium difficile toxin B enhances cytokine-mediated induction of NO synthase II.

In order to investigate the involvement of Ras and/or Rho proteins in the induction of the inducible isoform of nitric oxide synthase (NOS II) we used HMG-CoA reductase inhibitors (statins) and Clostridium difficile toxin B (TcdB) as pharmacological tools. Statins indirectly inhibit small G proteins by preventing their essential farnesylation (Ras) and/or geranylgeranylation (Rho). In contrast, TcdB is a glucosyltransferase and inactivates Rho-proteins directly. Human A549/8- and DLD-1 cells as well as murine 3T3 fibroblasts were preincubated for 18 h with statins (1 - 100 microM) or TcdB (0.01-10 ng ml(-1)). Then NOS II expression was induced by cytokines. NOS II mRNA was measured after 4 - 8 h by RNase protection assay, and NO production were measured by the Griess assay after 24 h. Statins and TcdB markedly increased cytokine-induced NOS II mRNA expression and NO production. Statin-mediated enhancement of NOS II mRNA expression was reversed almost completely by cotreatment with mevalonate or geranylgeranylpyrophosphate. It was only slightly reduced by farnesylpyrophosphate. Therefore, small G proteins of the Rho family are likely to be involved in NOS II induction. In A549/8 cells stably transfected with a luciferase reporter gene under the control of a 16 kb fragment of the human NOS II promoter (pNOS2(16)Luc), statins produced only a small increase in cytokine-induced NOS II promoter activity. In contrast, statins had a considerable superinducing effect in DLD-1 cells stably transfected with pNOS2(16)Luc. In conclusion, our studies provide evidence that statins and TcdB potentiate cytokine-induced NOS II expression via inhibition of small G proteins of the Rho family. This in turn results in an enhanced NOS II promoter activity and/or a prolonged NOS II mRNA stability.

Complex contribution of the 3'-untranslated region to the expressional regulation of the human inducible nitric-oxide synthase gene. Involvement of the RNA-binding protein HuR.

Cytokine stimulation of human DLD-1 cells resulted in a marked expression of nitric-oxide synthase (NOS) II mRNA and protein accompanied by only a moderate increase in transcriptional activity. Also, there was a basal transcription of the NOS II gene, which did not result in measurable NOS II expression. The 3'-untranslated region (3'-UTR) of the NOS II mRNA contains four AUUUA motifs and one AUUUUA motif, known to destabilize the mRNAs of proto-oncogenes, nuclear transcription factors, and cytokines. Luciferase reporter gene constructs containing the NOS II 3'-UTR showed a significantly reduced luciferase activity. The embryonic lethal abnormal vision (ELAV)-like protein HuR was found to bind with high affinity to the adenylate/uridylate-rich elements of the NOS II 3'-UTR. Inhibition of HuR with antisense constructs reduced the cytokine-induced NOS II mRNA, whereas overexpression of HuR potentiated the cytokine-induced NOS II expression. This provides evidence that NOS II expression is regulated at the transcriptional and post-transcriptional level. Binding of HuR to the 3'-UTR of the NOS II mRNA seems to play an essential role in the stabilization of this mRNA.

Comparative effects of several nitric oxide donors on intracellular cyclic GMP levels in bovine chromaffin cells: correlation with nitric oxide production.

1. Sodium nitroprusside, S-nitroso-N-acetyl-D,L-penicillamine, Spermine NONOate and DEA NONOate raised cyclic GMP levels in bovine chromaffin cells in a time and concentration dependent manner with different potencies, the most potent being DEA/NO with an EC50 value of 0.38 +/- 0.02 microM. 2. Measurements of NO released from these donors revealed that DEA/NO decomposed with a half-life (t1/2) of 3.9 +/- 0.2 min. The t1/2 for SPER/NO was 37 +/- 3 min. SNAP decomposed more slowly (t1/2 = 37 +/- 4 h) and after 60 min the amount of NO produced corresponded to less than 2% of the total SNAP present. The rate of NO production from SNAP was increased by the presence of glutathione. 3. For DEA/NO and SPER/NO there was a clear correlation between nitric oxide production and cyclic GMP increases. Their threshold concentrations were 0.05 microM and maximal effective concentration between 2.5 and 5 microM. 4. For SNAP, threshold activation was seen at 1 microM, whereas full activation required a higher concentration (500-750 microM). The dose-response for SNAP increases in cyclic GMP was shifted nearly two orders of magnitude lower in the presence of glutathione. At higher concentrations an inhibition of cyclic GMP accumulation was found. This effect was not observed with either the nitric oxide-deficient SNAP analogue or other NO donors. 5. Although NO-donors are likely to be valuable for studying NO functions, their effective concentrations and the amount of NO released by them are very different and should be assessed in each system to ensure that physiological concentrations of NO are used.

Phosphorylation of tyrosine hydroxylase by cGMP-dependent protein kinase in intact bovine chromaffin cells.

The phosphorylation of the enzyme tyrosine hydroxylase by the cGMP pathway was investigated in chromaffin cells from the bovine adrenal medulla. The nitric oxide donor, sodium nitroprusside, and the natriuretic peptide, C-type natriuretic peptide, which are able to increase cGMP levels and cGMP-dependent protein kinase activity, produced significant increases in the phosphorylation level of tyrosine hydroxylase in a time- and concentration-dependent manner. The pretreatment of the cells with the soluble guanylyl cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one blocked the effect of sodium nitroprusside. This result indicates that cGMP production by this enzyme mediated this effect. Experiments performed with a cGMP-dependent protein kinase inhibitor, the Rp-isomer of 8-(4-chlorophenylthio)-cyclic guanosine monophosphorothioate, which blocked the effects of both sodium nitroprusside and C-type natriuretic peptide, demonstrated that the phosphorylation increases evoked by both compounds were mediated by the activation of cGMP-dependent protein kinase. In cells incubated with the adenylyl cyclase activator, forskolin, an increase in the phosphorylation level of the tyrosine hydroxylase was also found. When cells were treated simultaneously with forskolin and sodium nitroprusside or C-type natriuretic peptide, an additive effect on tyrosine hydroxylase phosphorylation was not observed. This suggests that cAMP- and cGMP-dependent protein kinases may phosphorylate the same amino acid residues in the enzyme. Western blot analysis of soluble extracts from chromaffin cells detected specific immunoreactivity for two different commercial antibodies raised against cGMP-dependent protein kinase (both Ialpha and Ibeta isoforms). Electrophoretic mobility correlates with that of purified PKG Ialpha. Because the phosphorylation of the tyrosine hydroxylase correlates with increases in its enzymatic activity and thus with augmentation in the cell capacity to synthesize catecholamines, our results indicate that a cGMP-based second messenger pathway participates in catecholamine biosynthesis regulation in chromaffin cells, a mechanism which may be widespread in other catecholamine-synthesizing cells.

Cytokine induction of NO synthase II in human DLD-1 cells: roles of the JAK-STAT, AP-1 and NF-kappaB-signaling pathways.

1. In human epithelial-like DLD-I cells, nitric oxide synthase (NOS) II expression was induced by interferon-gamma (100 u ml(-1)) alone and, to a larger extent, by a cytokine mixture (CM) consisting of interferon-gamma, interleukin-1beta (50 u ml(-1)) and tumor necrosis factor-alpha (10 ng ml(-1)). 2. CM-induced NOS II expression was inhibited by tyrphostin B42 (mRNA down to 1%; nitrite production down to 0.5% at 300 microM) and tyrphostin A25 (mRNA down to 24%, nitrite production down to 1% at 200 microM), suggesting the involvement of janus kinase 2 (JAK-2). Tyrphostin B42 also blocked the CM-induced JAK-2 phosphorylation (kinase assay) and reduced the CM-stimulated STAT1alpha binding activity (gel shift analysis). 3. CM reduced the nuclear binding activity of transcription factor AP-1. A heterogenous group of compounds, that stimulated the expression of c-fos/c-jun, enhanced the nuclear binding activity of AP-1. This group includes the protein phosphatase inhibitors calyculin A, okadaic acid, and phenylarsine oxide, as well as the inhibitor of translation anisomycin. All of these compounds reduced CM-induced NOS II mRNA expression (to 9% at 50 nM calyculin A; to 28% at 500 nM okadaic acid; to 18% at 10 microM phenylarsine oxide; and to 19% at 100 ng ml(-1) anisomycin) without changing NOS II mRNA stability. In cotransfection experiments, overexpression of c-Jun and c-Fos reduced promoter activity of a 7 kb DNA fragment of the 5'-flanking sequence of the human NOS II gene to 63%. 4. Nuclear extracts from resting DLD-1 cells showed significant binding activity for transcription factor NF-kappaB, which was only slightly enhanced by CM. The NF-kappaB inhibitors dexamethasone (1 microM), 3,4-dichloroisocoumarin (50 microM), panepoxydone (5 microg ml(-1)) and pyrrolidine dithiocarbamate (100 microM) produced no inhibition of CM-induced NOS II induction. 5. We conclude that in human DLD-1 cells, the interferon-gamma-JAK-2-STAT1alpha pathway is important for NOS II induction. AP-1 (that is downregulated by CM) seems to be a negative regulator of NOS II expression. NF-kappaB, which is probably important for basal activity of the human NOS II promoter, is unlikely to function as a major effector of CM in DLD-1 cells.

Functional coupling of nitric oxide synthase and soluble guanylyl cyclase in controlling catecholamine secretion from bovine chromaffin cells.

This study was designed to evaluate whether the enzymes of the nitric oxide/cyclic-GMP pathway, nitric oxide synthase and soluble guanylyl cyclase, are functionally coupled in controlling catecholamine secretion in primary cultures of bovine chromaffin cells. In immunocytochemical studies, 80-85% of the tyrosine hydroxylase-positive chromaffin cells also possessed phenylethanolamine-N-methyltransferase, f1p4cating their capability to synthesize epinephrine. Immunoreactivity for neuronal-type nitric oxide synthase was found in over 90% of all chromaffin cells. Reverse transcription-polymerase chain reaction also demonstrated neuronal-type nitric oxide synthase messenger RNA. Immunoreactivity for soluble guanylyl cyclase was detectable in over 95% of chromaffin cells. Double-labeling immunofluorescence studies co-localized neuronal-type nitric oxide synthase and soluble guanylyl cyclase with tyrosine hydroxylase and phenylethanolamine-N-methyltransferase in the majority of chromaffin cells. Chromaffin cells possessed basal nitric oxide synthase activity which could be stimulated by acetylcholine and inhibited by NG-nitro-L-arginine methyl ester. Activation of soluble guanylyl cyclase by endogenously synthesized nitric oxide or the nitric oxide donor compound sodium nitroprusside was blocked by the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Catecholamine release and the increase in cytosolic Ca2+ concentration evoked by acetylcholine were enhanced by inhibitors of the endogenous nitric oxide/cyclic-GMP pathway such as NG-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor Rp-8-pCPT-cGMPS. These data indicate that chromaffin cells possess an autocrine nitric oxide/cyclic-GMP pathway tonically controlling the inhibition of catecholamine release.

Distribution of [3H]diadenosine tetraphosphate binding sites in rat brain.

The distribution of the diadenosine tetraphosphate high-affinity binding sites has been studied in rat brain by an autoradiographic method using [3H]diadenosine tetraphosphate as the ligand. The binding characteristics are comparable to those described in studies performed on rat brain synaptosomes. White matter is devoid of specific binding. The range of binding site densities in gray matter varies from 3 to 15 fmol/mg of tissue, exhibiting a widespread but heterogeneous distribution. The highest densities correspond to the seventh cranial nerve, medial superior olive, pontine nuclei, glomerular and external plexiform layers of the olfactory bulb, and the granule cell layer of the cerebellar cortex. Intermediate density levels of binding correspond to different cortical areas, several nuclei of the amygdala, and the oriens and pyramidal layers of the hippocampal formation. The localization of diadenosine tetraphosphate binding sites in the brain may provide information on the places where diadenosine polyphosphate compounds can be expected to function in the central nervous system.

Effect of cyclic GMP-increasing agents nitric oxide and C-type natriuretic peptide on bovine chromaffin cell function: inhibitory role mediated by cyclic GMP-dependent protein kinase.

Both sodium nitroprusside (SNP), a nitric oxide (NO) generator, and C-type natriuretic peptide (CNP) have been found to raise cGMP levels in bovine chromaffin cells in a time- and concentration-dependent manner. The effect of these compounds on catecholamine secretion and calcium influx has also been studied, and both compounds were found to produce a slowly developing inhibitory effect on acetylcholine- or depolarization-stimulated catecholamine secretion and calcium increases without affecting the spontaneous release or the basal intracellular Ca2+ concentration. These inhibitory effects were observed only at high doses of acetylcholine or high levels of extracellular potassium and required concentrations of SNP or CNP very similar to those that increased cGMP levels. Preincubation with 100 microM zaprinast, a cGMP-phosphodiesterase inhibitor able to increase cGMP levels, mimicked the inhibitory effects of SNP and CNP. We investigated the effect of the soluble guanylate cyclase inhibitor methylene blue and the cGMP-dependent protein kinase (PKG) inhibitor 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, Rp isomer, on inhibition by SNP or CNP. Although methylene blue (10 microM) partially prevented the inhibitory effect of SNP, it did not do so for that produced by CNP, thus indicating that SNP acts through cGMP produced by the NO-activated guanylate cyclase. 8-(4-Chlorophenylthio)-guanosine 3',5'-cyclic monophosphorothioate, Rp isomer totally reversed both the SNP and CNP inhibitory effects. These results suggest that the activation of PKG mediates the inhibition induced by SNP and CNP. We successfully measured the PKG activity from cells preincubated with SNP or CNP, and our results show that this enzymatic activity increased with a time dependence very similar to the increase in the cGMP levels. Our results indicate that NO and CNP peptide inhibit secretagogue-stimulated catecholamine release via activation of soluble and particulate isoforms of the guanylate cyclase, respectively, presumably by inhibition of calcium entry through voltage-activated calcium channels. This inhibitory effects seems to be mediated by activation of the PKG.

Activation of NO:cGMP pathway by acetylcholine in bovine chromaffin cells. Possible role of Ca2+ in the down-regulation of cGMP signaling.

The production of cyclic GMP (cGMP) induced by acetylcholine and other stimuli was studied in bovine chromaffin cells. Acetylcholine increased intracellular cGMP in a transitory (peak at 2 min) and concentration-dependent manner (estimated half maximal increase, EC50 = 61 +/- 5 microM). NG-nitro-L-arginine methyl ester (NAME) inhibited such a rise in cGMP with a half maximal inhibitory concentration (IC50) of 231 +/- 55 microM. The acetylcholine-induced increase in cGMP was also inhibited by a calmodulin antagonist (calmidazolium, 30 microM) and by the absence of extracellular calcium. Other agents that strongly increased cytosolic calcium concentration ([Ca2+]i) as acetylcholine did, such as the nicotinic-agonist, 1,1-dimethyl-4-phenylpiperazinium (DMPP), high-KCl (50 mM), and ionomycin, also caused a rise in cGMP in cultured bovine chromaffin cells. Veratridine, an activator of sodium channels, produced a slowly developing calcium increase and no significant cGMP production. The muscarinic-agonist, muscarine, failed to increase cytosolic calcium, and was the weakest stimulator of cGMP production. cGMP formation, induced by sodium nitroprusside (SNP, 100 microM) and by C-type natriuretic peptide (CNP, 100 nM), was inhibited by 30-40% by increasing [Ca2+]i with ionomycin. This inhibition was abolished by calmidazolium (30 microM) and by the absence of calcium in the extracellular medium. In conclusion, bovine chromaffin cells synthesize nitric oxide (NO) to activate guanylate cyclase in response to several stimuli, which increase [Ca2+]i. Moreover, the increase in [Ca2+]i also stimulates a Ca2+/calmodulin phosphodiesterase, which could down-regulate the levels of cGMP in these cells.

Cyclic GMP-dependent protein kinase activation mediates inhibition of catecholamines secretion and Ca2+ influx in bovine chromaffin cells.

The effects of the membrane-permeable cGMP analogue, 8-bromoguanosine 3':5'-cyclic monophosphate on acetylcholine-evoked catecholamine secretion and cytosolic calcium increases were studied in chromaffin cells from the bovine adrenal gland. Preincubation with 100 microM 8-bromoguanosine 3':5'-cyclic monophosphate during 10 and 30 min decreased the acetylcholine-evoked catecholamine release by 16 +/- 3% and 27 /+- 5%, respectively. The cytosolic calcium increases triggered by acetylcholine and 30 mM KCl were also inhibited by 30 min of preincubation with this compound by 27 +/- 4 and 34 /+- 12%, respectively. Changes in membrane potential induced by acetylcholine and KCl were not affected by preincubation with 8-bromoguanosine 3':5'-cyclic monophosphate. The cyclic GMP-dependent protein kinase inhibitor N-[2-(methylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride-at l micron abolished the inhibitory effect of 8-bromoguanosine 3':5'-cyclic monophosphate on acetylcholine-evoked calcium increase. By contrast, a potent and selective inhibitor against cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide did not block the 8-bromoguanosine 3':5'-cyclic monophosphate effect. Additionally, 8-bromoguanosine 3':5'-cyclic monophosphate stimulated histone F2b phosphorylation by a partial purified cGMP-dependent protein kinase from chromaffin cells. The extent of histone phosphorylation was reduced by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride and 8-(4-chlorophenylthio)-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer, a specific inhibitor against cyclic GMP-dependent protein kinase, whereas it was not modified by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide. The results suggest that the inhibitory effects of 8-bromoguanosine 3':5' cyclic monophosphate on chromaffin cells are mediated through the activation of cGMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of the dihydropyridine-insensitive Ca2+ influx by 8-bromo-guanosine-3':5'-monophosphate, cyclic (8-Br-cGMP) in bovine adrenal chromaffin cells.

Pretreatment of chromaffin cells with the permeable analogue of cGMP, 8-Br-cGMP (100 microM), leads to a reduction (35%) of depolarization-evoked intracellular calcium concentration ([Ca2+]i) increases. There is evidence that bovine adrenal chromaffin cells are provided with both dihydropyridine-sensitive and -resistant voltage-sensitive Ca2+ influx pathways. Combined incubations with nifedipine 10 microM and 8-Br-cGMP reduced KCl-evoked intracellular Ca2+ concentration to a greater extent that each compound separately. Moreover, 8-Br-cGMP failed to affect the [Ca2+]i transient induced by the L-type Ca2+ channel agonist Bay K 8644 (1 microM) under conditions of low depolarization. Neomycin (0.2 mM) and omega-AgaToxin-IVA (AgTx) (1 microM) inhibited the calcium transient to a similar extent, and this inhibition was not enhanced by the presence of 8-Br-cGMP. It is concluded that 8-Br-cGMP modulated the dihydropyridine-insensitive Ca2+ influx pathway in the chromaffin cell.

8-Azido-adenine nucleotides as substrates of ecto-nucleotidases in chromaffin cells: inhibitory effect of photoactivation.

The components of the ecto-nucleotidase pathway at the extracellular surface of adrenal chromaffin cells are the enzymatic activities responsible for the hydrolysis of granular nucleotide compounds released during the secretory response. The azido-nucleotides have been largely employed to characterize nucleotide binding sites. The 8-azido-adenine nucleotides were studied as substrates of ecto-nucleotidases in cultured chromaffin cells by HPLC procedures. 8-Azido-ATP (8-N3-ATP) was a good substrate for ecto-ATPase activity, the Km value was 256.30 +/- 36.41 microM, and the Vmax value was 14.33 +/- 0.84 nmol/min x 10(6) cells. 8-Azido-ADP (8-N3-ADP) was dephosphorylated by the ecto-ADPase activity with a Km value of 595.29 +/- 67.44 microM and Vmax value of 6.86 +/- 0.45 nmol/min x 10(6) cells. These kinetic parameters were similar to those obtained with ATP and ADP in the same culture and incubation conditions. 8-Azido-AMP (8-N3-AMP) was not hydrolyzed by the ecto-5'-nucleotidase activity. The 8-azido-nucleotides competitively inhibited the hydrolysis of adenine nucleotides, with Ki values in the same range as the Km. After uv photoactivation, the three 8-azido-nucleotides (100 microM) irreversibly inhibited and to a similar extent, between 40 and 55%, each of ecto-nucleotidase activities. UV photoactivation in the presence of nucleotides in the same concentration range was an effective protection from the inhibition.

Extracellular hydrolysis of diadenosine polyphosphates, ApnA, by bovine chromaffin cells in culture.

An ectoenzyme hydrolyzing diadenosine polyphosphates (ApnA) to AMP and Ap(n-1) has been studied in cultured chromaffin cells from bovine adrenal medulla. The KM value for extracellular Ap4A hydrolysis was 2.90 +/- 0.72 microM, the V(max) value obtained was 11.59 +/- 0.92 pmol/min x 10(6) cells (116 pmol/min.mg total protein). Ap3A, Ap5A, Ap6A, and Gp4G were competitive inhibitors of Ap4A hydrolysis with K(i) values of 3.65, 1.10, 1.20, and 2.65 microM, respectively. Phosphatidylinositol-specific phospholipase C removes the ApnA hydrolase activity from cultured chromaffin cells, suggesting an anchorage of this protein to the plasma membrane through the phosphatidylinositol. The turnover time for this enzyme calculated in the presence of cycloheximide was 38.94 +/- 1.53 hr for cultured chromaffin cells.