Biochemical activity and multiple locations of particulate guanylate cyclase in Rhyacophila dorsalis acutidens (Insecta: Trichoptera) provide insights into the cGMP signalling pathway in Malpighian tubules.

In insect renal physiology, cGMP and cAMP have important regulatory roles. In Drosophila melanogaster, considered a good model for molecular physiology studies, and in other insects, cGMP and cAMP act as signalling molecules in the Malpighian tubules (MTs). However, many questions related to cyclic nucleotide functions are unsolved in principal cells (PC) and stellate cells (SC), the two cell types that compose the MT. In PC, despite the large body of information available on soluble guanylate cyclase (sGC) in the cGMP pathway, the functional circuit of particulate guanylate cyclase (pGC) remains obscure. In SC, on the other side, the synthesis and physiological role of the cGMP are still unknown. Our biochemical data regarding the presence of cyclic nucleotides in the MTs of Rhyacophila dorsalis acutidens revealed a cGMP level above the 50%, in comparison with the cAMP. The specific activity values for the membrane-bound guanylate cyclase were also recorded, implying that, besides the sGC, pGC is a physiologically relevant source of cGMP in MTs. Cytochemical studies showed ultrastructurally that there was a great deal of pGC on the basolateral membranes of both the principal and stellate cells. In addition, pGC was also detected in the contact zone between the two cell types and in the apical microvillar region of the stellate cells bordering the tubule lumen. The pGC signal is so well represented in PC and, unexpectedly in SC of MTs, that it is possible to hypothesize the existence of still uncharacterized physiological processes regulated by the pGC-cGMP system.

Interleukin-1beta induces apoptosis in GL15 glioblastoma-derived human cell line.

Interleukin 1-beta (IL-1beta) induces apoptosis in a glioblastoma-derived human cell line, exhibiting a poorly differentiated astrocytic phenotype. The apoptotic effect was demonstrated by analyzing nuclear morphology, in situ DNA fragmentation, and by ELISA detection of cytoplasmatic nucleosomes. We correlated the degree of differentiation of GL15 cells with the apoptotic response: 1) 4',6-diamidino-2-phenylindole staining, combined with glial fibrillary acidic protein (GFAP) immunofluorescence, showed that the cells with apoptotic nuclei express low levels of GFAP; and 2) at 13 days of subculture, in a more differentiated state, GL15 cells did not respond with apoptosis to IL-1beta. In this cell line, nonrandom chromosome changes and the expression of SV40 early region have been previously shown. The involvement of p42/p44 mitogen-activated protein kinase (MAPK) pathway in the induction of apoptosis by IL-1beta was hypothesized. Previous studies have shown that SV40 small T antigen partially inhibits phosphatase 2A, leading to an enhancement of the steady-state activity of p42/p44 MAPK pathway. PD-098059, specific inhibitor of p42/p44 MAPK pathway, counteracts the apoptotic effect of IL-1beta, whereas SB-203580, specific inhibitor of p38 stress-activated protein kinase (SAPK) pathway, is ineffective. The imbalance between MAPK and SAPK pathways has been proposed as a key factor in determination of cell fate. Our results demonstrate that a further stimulation of p42/p44 MAPK pathway can constitute a death signal in tumor cells in which genomic damage and MAPK pathway control alterations occur.

Ultracytochemical localization of guanylate cyclase after stimulation with natriuretic peptides in frog choroid plexus.

The ultracytochemical localization of particulate guanylate cyclase (GC) has been studied in Rana esculenta choroid plexus after activation with rat atrial natriuretic peptide (ANP), porcine brain natriuretic peptide (BNP) and porcine C-type natriuretic peptide (CNP). This study shows that the three peptides are activators of GC in the choroid plexus as demonstrated by the presence of reaction products at the level of the epithelium and sinusoids. In the apical zone of the epithelial cells predominantly BNP seems to activate GC, while ANP and CNP activate GC mainly at the level of the lateral surfaces. Moreover, ANP stimulates the enzyme along the basal membrane of the epithelial cells as well as the membranes of the endothelium of the sinusoids. In the presence of CNP, enzyme stimulation can also be found at the level of the endocellular membranes. These results confirm that the choroid plexus is an organ with receptors for the natriuretic peptides which are involved in the processes of osmoregulation and the control of cerebrospinal fluid production.

Biochemical and cytochemical analyses of BNP-stimulated guanylate cyclase in frog choroid plexus.

This study shows that in the choroid plexus of Rana esculenta particulate guanylate cyclase (GC) is appreciably stimulated by porcine brain natriuretic peptide (BNP). Ultracytochemical tests for GC show that BNP notably increases the enzymatic reaction product along the apical surfaces of the epithelial cells. It can therefore be hypothesized that the apical zone of the epithelial cells possess receptors which have a particular affinity for BNP produced in the central nervous system and dumped into the cerebrospinal fluid. These results, together with those of a previous study [32], confirm that the choroid plexus is an organ which has receptors for the natriuretic peptides which are involved in the processes of osmoregulation and the control of cerebrospinal fluid production.

Response of guanylate cyclase to atrial natriuretic factor in epithelial cells of the frog choroid plexus.

This study shows that the choroid plexus of Rana esculenta contains a guanylate cyclase particulate (GCp), similar to that identified in Mammalia, that is quite sensitive to the atrial natriuretic factor (ANF). The cytochemical tests for GCp show that ANF increases the enzymatic reaction products. Deposits are observed on the apical portion, at the basal level and along the lateral edges of the epithelial cells, with the exclusion of some intercalary epithelial cells with reaction-lacking microvilli. In particular, ANF seems to intensely stimulate the GCp activity along the lateral membranes of the epithelial cells delimiting enlarged intercellular spaces, which are probably dilated for the transport of water and solutes. These data confirm the osmoregulatory role of the hormone and its control of cephalorachidian liquid composition.

Activity trends of adenylate and guanylate cyclases and cAMP and cGMP levels during the first embryonic stages of Bufo bufo.

1. During the first stages of embryonic development of Bufo bufo, the levels of cAMP and cGMP showed interesting opposite trends analogous to the trends of their respective enzymes. 2. In the late segmentation, the high increase of guanylate cyclase specific activity and the corresponding rise of embryonic level of cGMP, could indicate the involvement of this nucleotide in cellular proliferation. 3. During the following stage of the gastrulation, the increase of adenylate cyclase specific activity, coupled to the loss of guanylate cyclase specific activity, could suggest the importance of cAMP in the phenomena of differentiation induction. 4. Furthermore, the cytochemical investigation of adenylate and guanylate cyclase localization seems to confirm the prominent role of cAMP during the differentiation phases.

Acid phosphatases in the frog (Rana esculenta) skeletal muscle. Purification and some properties of the low molecular weight enzyme.

1. The presence of high-Mr and low-Mr acid phosphatases [orthophosphoric-monoester phosphohydrolase, (acid optimum), EC 3.1.3.2] in the skeletal muscle of frog Rana esculenta was reported. 2. The subcellular localization and some characteristics of both enzymes were also described. 3. The low-Mr AcPase was purified to homogeneity. The enzyme did not absorb on Concanavalin A-Sepharose 4B indicating that this was not a glycoprotein. 4. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr 13.7 +/- 0.8 kDa in the presence of sodium dodecyl sulphate. 5. The Mr of the native enzyme was 14.0 +/- 1.1 kDa as determined by gel filtration on a Sephadex G-100 column. The isoelectric point was 6.02. 6. The enzyme was strongly inhibited by 1 mM Ag+, Hg2+, Sn2+ and Cu2+ while other cations both at 10(-2) and 10(-3) M showed little or no effect. 7. The enzyme was insensitive to NaF and tartrate but was strongly deactivated by formaldehyde, PMB, Iodoacetamide and Triton X-100. Phosphate was a competitive inhibitor (k1 = 0.83 mM). 8. The best substrate for the enzyme was p-nitrophenylphosphate but phenylphosphate, flavin mononucleotide and o-P-tyrosine were also hydrolyzed, though at different rates. 9. The enzyme activity was enhanced in the presence of methanol, ethanol, acetone and glycerol indicating a phosphotransferase activity.

Cytochemical and biochemical observations on the alveolar guanylate cyclase of golden hamster lung.

Particulate guanylate cyclase (GTP pyrophosphate-lyase (cyclizing] has been cytochemically evidentiated in the cells which make-up the lung air-blood barrier. The cytochemical procedure utilized demonstrates the presence of membrane-bound guanylate cyclase activity through precipitation of lead pyrophosphate in tissues incubated with GTP or with guanylyl imidodiphosphate. Electron microscopic examination reveals that guanylate cyclase (GC) is localized, as micropinocytic vesicles, within endothelial components of small blood vessels, in basal lamina and in the flat alveolar cells. The secretory alveolar cells also exhibit the positive GC reactivity in their peripheric cytoplasm and in their microvilli. The observations support that GC and cGMP are involved in cellular transport phenomena. The enzyme might play a role in the secretion process of surface active material. Positive staining has been found also in other types of cells, namely alveolar macrophages and fibroblasts. A biochemical evaluation of GC activity shows that about 30-40% of this activity is associated with the particulate fraction, which justifies its abundance in the cytochemical reports shown in the paper.

Cyclic nucleotides and haemoglobin concentration in rabbit bone marrow cell suspensions stimulated by purified erythropoietin.

This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db-cAMP) or dibutyryl cGMP (db-cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml-1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml-1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db-cAMP or db-cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.

Evidence for an inotropic positive action of cGMP during excitation-contraction coupling in frog sartorius muscle.

Within the realm of the general hypothesis concerning the role of cGMP on intracellular calcium regulation in biological systems, we have investigated the action of cyclic nucleotides during excitation-contraction coupling in frog sartorius muscle. Our data show that several guanosine nucleotides (GTP, GDP, dibutyryl-cGMP) can increase the isometric twitch tension with a maximum increase of 40% in the muscles treated with cGMP. This increase is completely independent of external Ca2+ concentration. The use of dantrolene sodium (known to inhibit calcium release from sarcoplasmic reticulum) results in a decrease in the twitch tension with a contemporary decrease in the intracellular levels of cGMP; whereas, the addition of cGMP to the muscles treated with dantrolene antagonizes, at least partially, the effect of the drug on tension development. Finally, in chemically skinned muscles, cGMP induces a reversible contracture equal to approximately one-half of that evoked by 10(-4) M Ca2+.