Formaldehyde fixation of cGMP in distinct cellular pools and their recognition by different cGMP-antisera. An immunocytochemical study into the problem of serum specificity

Three different antisera raised against the same formaldehyde fixed cGMP conjugate were tested for their specificity in two non-biological and two biological model systems. The first non-biological model system was based on nucleotides fixed to gelatin by formaldehyde and the other non-biological model was nitrocellulose paper as a carrier for nucleotides coupled to proteins by formaldehyde. All antisera proved specific for cGMP in both models. As biological models we used the in vitro incubated hippocampus slice and the in vitro incubated aortic ring. In hippocampus slices all three antisera showed cGMP-producing cells after atrial natriuretic factor stimulation. However, there were significant differences in the visualization of cGMP-immunoreactivity between the three antisera when sodium nitroprusside or potassium were used to stimulate cGMP production. Nevertheless, these differential staining patterns all showed cGMP-immunoreactivity using the conventional immunocytochemical control tests. In the aorta ring all three antisera showed the same strong increase in cGMP-immunoreactivity after in vitro stimulation with sodium nitroprusside. These results were corroborated by biochemical assay of cGMP. We conclude that these three antisera all demonstrate cGMP-immunoreactivity in the biological models used. The different staining patterns that occur are caused by differences in the microchemical milieu of the formaldehyde-fixed cGMP. The use of different antibodies to cGMP may give information about this microchemical milieu which may eventually contribute to a better understanding of different intracellular cGMP pools.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation

Summary Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O₂-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O₂-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10^–5 M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced. The synthetic analog of atrial natriuretic peptide, atriopeptin III (10^–7 M), greatly elevated cGMP immunoreactivity in the type I cells. On the other hand, sodium nitroprusside (1 mM) elevated cGMP staining mostly in vascular elements of the carotid body in vitro. The data implicate the involvement of cyclic nucleotides in transduction of natural chemosensory stimuli by the type I cells in rat carotid body.     

Requirement for bivalent cations in the actions of insulin and sodium nitroprusside on metabolism in rat adipocytes

The requirement for Ca2+ and Mg2+ in the actions of insulin and sodium nitroprusside on rat adipocyte metabolism was investigated: sodium nitroprusside, but not insulin, increased cGMP levels in cells incubated in the absence of Ca2+ and/or Mg2+; sodium nitroprusside and insulin are unable to increase the incorporation of [14C]glucose into triglycerides and [14C]leucine into proteins in the absence of Ca2+ and Mg2+; sodium nitroprusside and insulin showed antilipolytic actions in Ca2+- and Mg2+-free medium. We conclude that in the absence of Ca2+ and Mg2+, sodium nitroprusside and insulin have very similar regulatory properties on triglyceride, protein synthesis and adrenaline-stimulated lipolysis, but not on cGMP levels in rat adipocytes. This could provide evidence that omission of bivalent cations was inhibitory at more than one site, or that sodium nitroprusside mimics insulin's actions by another mechanism that does not involve cGMP.     

Immunocytochemical localization of cAMP and cGMP in cells of the rat carotid body following natural and pharmacological stimulation.

Although the chemoreceptive function of the carotid body has been known for many decades, the cellular mechanisms of sensory transduction in this organ remain obscure. Common elements in the transductive processes of many cells are the cyclic nucleotide second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Studies from our laboratory have revealed stimulus-induced changes in cyclic nucleotide levels in the carotid body as measured by RIA, but such changes in second messenger levels have not been localized to specific cellular elements in the organ. The present immunocytochemical study utilized the avidin-biotin-peroxidase method to investigate the distribution of cAMP and cGMP in the rat carotid body and to assess changes in the intensity of immunostaining following in vitro stimulation by hypoxia, forskolin, sodium nitroprusside, high potassium, and atrial natriuretic peptide. Both cAMP and cGMP immunoreactivity were localized to type I cells of organs maintained in vivo and fixed by perfusion. Organs exposed to 100% O2-equilibrated media in vitro produced low but visible levels of cAMP immunoreactivity in a majority of type I cells; hypoxia (5% O2-equilibrated media) for 10 min moderately increased the level of immunoreactivity; forskolin (10(-5) M), or forskolin combined with hypoxia, dramatically increased cAMP levels in virtually all cells. Moderate levels of cGMP immunoreactivity in control carotid bodies in vitro were strikingly reduced by hypoxia; a significant increase in cGMP levels occurred following incubation in high potassium (100 mM), and under these conditions, the decrease in cGMP immunoreactivity with hypoxia was much more pronounced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biochemical mechanisms of atrial natriuretic factor action

Since atrial natriuretic factor (ANF) is a natriuretic and vasodilatory hormone, its mechanisms of action expectedly involve so-called negative pathways of cell stimulation, notably cyclic nucleotides. Indeed, the guanylate cyclase-cyclic GMP (cGMP) system appears to be the principal mediator of ANF's action. Specifically, particulate guanylate cyclase, a membrane glycoprotein, transmits ANF's effects, as opposed to the activation of soluble guanylate cyclase such agents as sodium nitroprusside. The stimulation of particulate guanylate cyclase by ANF manifests several characteristics. One of them is the functional irreversibility of stimulation with its apparent physiological consequences: the extended impact of ANF on diuresis and vasodilation in vivo lasts beyond the duration of increased plasma ANF levels and is accompanied by a prolonged elevation of cGMP. Another characteristic is the parallelism between guanylate cyclase stimulation and increases of cGMP in extracellular fluids. cGMP egression appears to be an active process, yet its physiological implications remain to be uncovered. In heart failure, cGMP continues to reflect augmented ANF levels, suggesting that in this disease, the lack of an ANF effect on sodium excretion is due to a defect distal to cGMP generation. In hypertension, where ANF levels are either normal or slightly elevated, probably secondary to high blood pressure, the ANF responsiveness of the particulate guanylate cyclase-cGMP system, the hypotensive effects, diuresis and natriuresis are exaggerated. The implications of this exaggerated responsiveness of the ANF-cGMP system in the pathophysiology of hypertension and its potential therapeutic connotations remain to be evaluated.     

Twenty-four-hour variation of L-arginine/nitric oxide/cyclic guanosine monophosphate pathway demonstrated by the mouse visceral pain model

The L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L-arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light-dark cycle. Animals were injected s.c. with saline, 2 mg/kg L-arginine (a NO precursor), 75 mg/kg L-N(G)-nitroarginine methyl ester (L-NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p-benzoquinone (PBQ)-induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L-arginine or L-NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time-dependent differences in PBQ-induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L-NAME, methylene blue, or sodium nitroprusside, but not saline or L-arginine. These results suggest that components of L-arginine/NO/cGMP pathway exhibit biological time-dependent effects on visceral nociceptive process.     

Inhibition of cyclic GMP hydrolysis in human corpus cavernosum smooth muscle cells by vardenafil, a novel, selective phosphodiesterase type 5 inhibitor.

One of the key mediators of penile erectile function is nitric oxide (NO), which activates soluble guanylyl cyclase within the smooth muscle of erectile tissue and stimulates the production of cGMP. In addition to synthesis by cyclases, intracellular cGMP concentrations are tightly regulated by phosphodiesterases, which hydrolyze and inactivate cyclic nucleotides. In this study, we compared the inhibition of cGMP hydrolysis by vardenafil and sildenafil; two inhibitors selective for phosphodiesterase type 5 (PDE5). Vardenafil is a novel, high affinity PDE5 inhibitor currently under clinical development. In soluble extracts of human corpus cavernosum smooth muscle cells, vardenafil and sildenafil effectively inhibited cGMP hydrolysis at substrate concentrations of 1, 5 and 10 microM cGMP. The IC50 values for vardenafil were approximately 5-fold lower than for sildenafil at the substrate concentrations tested. Dixon plot analyses of the inhibition data demonstrated that vardenafil had a smaller inhibition constant (Ki = 4.5 nM) than sildenafil (Ki = 14.7 nM) in the same cellular extracts. In intact cells, 10 microM of the nitric oxide donor sodium nitroprusside resulted in a minimal (17%) increase in cGMP, relative to basal levels (321 +/- 65 fmol/mg prot). Treatment of cells with 10, 50 or 100 nM vardenafil, in the presence of 10 microM sodium nitroprusside, elevated cGMP levels in a dose dependent fashion, from 63% to 137% of basal levels. Equimolar concentrations of sildenafil also caused dose dependent increases in intracellular cGMP, but to a lesser extent (27-60%). These observations suggest that vardenafil is a more potent PDE5 inhibitor, than sildenafil in vitro. The more pronounced increase of cGMP in the presence of NO in intact cells suggests that vardenafil will be effective at lower doses than sildenafil under clinical conditions.     

Chemosensory behaviour and ciliary cyclic GMP-dependent protein kinase in Tetrahymena thermophila

The role of protein kinase, in particular cyclic GMP-dependent protein kinase (PKG), in the control of chemotaxis was studied in Tetrahymena thermophila using the membrane-permeable cGMP analogue 8-bromo-cGMP and the NO-generator sodium nitroprusside (SNP) that stimulates cGMP production by activating guanylate cyclase. Stimulation of chemoattraction was observed in the presence of 8-bromo-cGMP and nitroprusside when used in 10–100 μM concentrations in vivo. In vitro stimulation of ciliary membrane PKG activity was observed when using similar concentrations of cGMP or 8-bromo-cGMP to those in the in vivo experiments. In contrast, the protein kinase flavonol inhibitors quercitin and kaempherol block chemoattraction and reduce ciliary membrane PGK activity in vitro. For the inhibition of PKG, the IC-50 s for quercitin and kaempherol are 22 and 19 μM, respectively. The results suggest a modulating function of PKG on adaptory processes in cilia-mediated chemotaxis.

The ciliary membrane-associated PKG was partially characterized. Without added external protein kinase substrate in vitro, an endogenous ciliary membrane kinase activity showed phosphorylation of 55 and 97 kDa Triton-X-100 soluble proteins when analyzed by SDS-PAGE under reducing conditions and with ³²P-γ-ATP as phosphorylation donor. Phosphoamino acid analysis of PKG-phosphorylated proteins showed ³²P-phosphate labeling of serine and threonine residues. Ciliary membrane-associated PKG was further purified by carboxy-methyl-sephadex-column chromatography. The membrane enzyme was Mg²⁺⁺-dependent and had a pH optimum at 6.4. The carboxy-methyl-sephadex-eluted PKG was analyzed by electrophoresis on sodium dodecyl sulphate polyacrylamide gels showing a molecular weight of 70–75 kDa.     

Hydralazine decreases sodium nitroprusside-induced rat aortic ring relaxation and increased cGMP production by rat aortic myocytes

Association of hydralazine with nitrova-sodilators has long been known to be beneficial in the vasodilator treatment of heart failure. We previously found that hydralazine appeared to reduce the increase in cGMP induced by sodium nitroprusside in cultured rat aortic myocytes. In order to further explore this seemingly paradoxical interaction, we extended our initial observations in rat aortic myocytes and also determined the influence of hydralazine on sodium nitroprusside-induced relaxation of rat aortic rings. Hydralazine produced a concentration-dependent inhibition of sodium nitroprusside stimulation of cGMP production and caused a rightward shift of concentration-relaxation curves in aortic rings. A possible mechanism of the hydralazine-nitroprusside interaction could be the interference with bioactivation of the nitro-vasodilator to release nitric oxide. Recent evidence indicates that vascular NADH oxidase, an enzyme known to be inhibited by hydralazine, could be involved in this process. Accordingly, hydralazine was found to inhibit NADH oxidase activity in rat aortic myocytes at concentrations similar to those reducing sodium nitroprusside responses. It was concluded that antagonism of sodium nitroprusside action by hydralazine could be a consequence of interference with bioactivation of the former, apparently through inhibition of vascular NADH oxidase.     

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.     

In vivo and in vitro studies of a putative inhibitor of cyclic guanosine 3',5'-monophosphate production.

Our main objective was to test the efficacy of 6-anilino-5,8-quinolinedione (LY83583) in vivo, a putative inhibitor of cyclic guanosine 3',5'-monophosphate (cGMP) production. If the drug proved capable of lowering plasma, vascular, and kidney levels of cGMP and of inhibiting the hypotensive effect of sodium nitroprusside and methacholine, then LY83583 could be of potential use in exploring the contribution of cGMP to cardiovascular and renal physiology. We found that when administered to trained conscious rats, LY83583 (1-mg/kg bolus, followed by a 2-hr infusion of 3 mg/kg.hr) decreased plasma cGMP concentration by 36% (P less than 0.01). Doubling the dosage of drug (2-mg/kg bolus, 6 mg/kg.hr) decreased plasma cGMP by 46% (P less than 0.05). We next measured tissue levels of cGMP ex vivo from rats that had received LY83583 or vehicle for 2 hr. The cGMP content of aortic segments when LY83583 was infused at the low dose, or renal cortical tissue when LY83583 was infused at both doses, was not significantly different from the cGMP content of tissue from rats that had received vehicle. LY83583 in doses up to 10-mg/kg bolus, followed by 6 mg/kg.hr infusion also failed to attenuate the hypotensive response to sodium nitroprusside or methacholine in conscious rats. Last, we tested whether, in our hands, LY83583 could reduce cGMP of aortic segments and kidney cortical slices in vitro. We found that after 10 min of incubation, 10(-5) M LY83583 decreased intracellular cGMP by approximately 65% and 50% in aortic and kidney tissues, respectively. In order to ascertain whether LY83583 lowered cGMP by stimulating phosphodiesterase, we incubated tissues with 10(-4) M 3-isobutyl-1-methylxanthine to inhibit the enzyme. In the presence of 3-isobutyl-1-methylxanthine LY83583 still exerted an inhibitory effect on cGMP production by aortic and kidney tissues. In conclusion, although LY83583 is a useful agent to lower renal and vascular tissues levels of cGMP in vitro, its efficacy in vivo seems doubtful.     

Potentiation of NO-dependent activation of soluble guanylate cyclase by polyamines

The influence of polyamines (putrescine, spermidine, and spermine) on the activity of human platelet soluble guanylate cyclase and the stimulation of the enzyme by sodium nitroprusside (SNP), YC-1 and their combination was investigated. All these polyamines stimulated the guanylate cyclase activity and potentiated its activation by sodium nitroprusside. The stimulatory effects of sodium nitroprusside and putrescine (or spermine) were addidive; spermidine produced a synergistic activation and increased the additive effect. All the polyamines inhibited the enzyme activation by YC-1 and decreased the synergistic activation of SNP-stimulated guanylate cyclase activity by YC-1 with nearly the same potency. The ability of the investigated polyamines to potentiate and to increase synergistically (similar to to YC-1, but less effective) NO-dependent activation of soluble guanylate cyclase represents a new biochemical effect of these compounds; this effect should be taken into consideration, especially due to the endogenous nature of polyamines. The data obtained suggest, that specific biological functions of polyamines in the processes of growth and differentiation of cells may be also related to the ability of compounds to activate soluble guanylate cyclase and to increase intracellular cGMP level.     

Comparative Study of Mechanisms of Relaxation of Smooth Muscles in the Rat Bronchus and Pulmonary Artery

To compare mechanisms of relaxation of the vascular and bronchial smooth muscles that have different structural and functional organization and embryonic origin, some links were studied of the intracellular systems transducing signals of dilation to the smooth-muscular cellular apparatus in the rat main pulmonary artery and main bronchus. The pulmonary artery and bronchus relaxation was measured by change in the value of isometric contraction of isolated ring segments of the artery and bronchus under the effect of isoproterenol, 3-isobutyl-1-methylxanthine, forskolin, sodium nitroprusside, verapamil, adenosine, and papaverine. The results obtained have shown heterogeneity of the responses of the rat arterial and bronchial smooth muscles to action of relaxants. The vascular smooth muscle turned out to be more reactive: all used substances relaxed it, although to a different extent (from 30 to 100% ). In the bronchial smooth muscle, the same response, although less pronounced, was found for 4 out of 7 substances. The relaxation produced by stimulation of -adrenoreceptors turned out to be functionally more important for the pulmonary artery than for the bronchus. Nevertheless, the results obtained have shown that apart from the heterogeneity of the responses to external stimuli and involvement of different signal systems, there are the links of intracellular transduction systems that are equally involved in relaxation of the vascular and bronchial muscles. They proved to be intracellular systems of cyclic nucleotides, cAMP and cGMP.     

Vasodilator-stimulated protein phosphorylation in platelets is mediated by cAMP- and cGMP-dependent protein kinases

Vasodilators such as sodium nitroprusside, nitroglycerin and various prostaglandins are capable of inhibiting platelet aggregation associated with an increase of either cGMP or cAMP. In our studies with intact platelets, prostaglandin E1 and sodium nitroprusside stimulated the phosphorylation of several proteins which could be distinguished from proteins known to be phosphorylated by a calmodulin-regulated protein kinase or by protein kinase C. Prostaglandin E1 (10 microM) or dibutyryl cAMP (2 mM) stimulated the phosphorylation of proteins with apparent relative molecular masses, Mr, of 240,000, 68,000, 50,000, and 22,000 in intact platelets. These proteins were also phosphorylated in response to low concentrations (1-2 microM) of cAMP in a particulate fraction of platelets. In intact platelets, sodium nitroprusside (100 microM) and the 8-bromo derivative of cGMP (2 mM) increased the phosphorylation of one protein of Mr 50,000 which was also phosphorylated in response to low concentrations (1-2 microM) of cGMP in platelet membranes. An additional protein (Mr 24,000) appeared to be phosphorylated to a lesser degree in intact platelets by prostaglandin E1 and sodium nitroprusside. Since the phosphorylation of the protein of Mr 50,000 was stimulated both in intact platelets by cyclic-nucleotide-elevating agents and cyclic nucleotide analogs, as well as in platelet membranes by cyclic nucleotides, this phosphoprotein was analyzed by limited proteolysis, tryptic fingerprinting and phosphoamino acid analysis. These experiments indicated that the 50-kDa proteins phosphorylated by sodium nitroprusside and prostaglandin E1 were identical, and that the peptide of the 50-kDa protein phosphorylated by both agents was also the same as the peptide derived from the 50-kDa protein phosphorylated in platelet membranes by cGMP- and cAMP-dependent protein kinases, respectively. Regulation of protein phosphorylation mediated by cAMP- and cGMP-dependent protein kinases may be the molecular mechanism by which those vasodilators, capable of increasing either cAMP or cGMP, inhibit platelet aggregation.     

Calmodulin antagonists inhibit dihydropyridine calcium channel activator (BAY-K-8644) induced cGMP synthesis in pituitary tumor cells

The dihydropyridine calcium channel activator, BAY-K-8644, stimulates cGMP formation in ACTH-secreting mouse AtT-20 clonal corticotrophs. The recent report that calmodulin antagonists could inhibit dihydropyridine binding in several tissues suggested that these agents might also affect the cyclic nucleotide response to BAY-K-8644. In fact, TMB-8, trifluoperazine, and melittin, described as in vitro antagonists of calmodulin-dependent enzyme activities, all inhibited BAY-K-8644 induced cGMP synthesis in a concentration-dependent manner. The antagonists had no effect on cGMP formation stimulated by sodium nitroprusside or sodium azide. The calcium channel antagonist, nifedipine, did not stimulate cGMP formation nor did it alter the effect of BAY-K-8644 on accumulation of the nucleotide; one explanation thus is that the cyclase involved in cGMP formation is coupled to a low affinity binding site for BAY-K-8644, which is less accessible to other dihydropyridines. The relation of cyclic GMP formation to the function of the calcium channel in AtT-20 cells remains unknown.     

Understanding microwave-stimulated Romanowsky-Giemsa staining of plastic embedded bone marrow

Summary Bone marrow smears were made and fixed in methanol or formaldehyde. Marrow sections of various thicknesses were also prepared from formaldehyde fixed marrows embedded in paraffin or plastic (glycol methacrylate). The different smears and sections were then stained by a Romanowsky-Giemsa procedure. Some specimens were stained using a standard microwave-stimulated method previously used diagnostically. The effects of technical variations were studied, including degree of microwave irradiation and the staining time. Comparisons of the resulting staining outcomes showed that microwave stimulated Romanowsky-Giemsa staining of plastic sections is a rate controlled process. Unusual aspects of the staining pattern of plastic sections (namely the purple basophilic cytoplasms and nucleoli, and blue chromatin) are due to microwave stimulation and formaldehyde fixation respectively.     

Effects of exogenous donor of nitric oxide-sodium nitroprusside on energy production of rat reticulocytes

The effects of the sodium nitroprusside (SNP), a nitric oxide (NO) donor clinically used in the treatment of hypertensive emergencies on the energy production of rat reticulocytes were investigated. Rat reticulocyte-rich red blood cell suspensions were aerobically incubated without (control) or in the presence of different concentrations of SNP (0.1, 0.25, 0.5, 1.0 mM). SNP decreased total and coupled, but increased uncoupled oxygen consumption. This was accompanied by the stimulation of glycolysis, as measured by increased glucose consumption and lactate accumulation. Levels of all glycolytic intermediates indicate stimulation of hexokinase-phosphofructo kinase (HK-PFK), glyceraldehyde 3-phosphate dehydrogenase (GAPD) and pyruvate kinase (PK) activities in the presence of SNP. Due to the decrease of coupled oxygen consumption in the presence of SNP, ATP production via oxidative phosphorylation was significantly diminished. Simultaneous increase of glycolytic ATP production was not enough to provide constant ATP production. In addition, SNP significantly decreased ATP level, which was accompanied with increased ADP and AMP levels. However, the level of total adenine nucleotides was significantly lower, which was the consequence of increased catabolism of adenine nucleotides (increased hypoxanthine level). ATP/ADP ratio and adenylate energy charge level were significantly decreased. In conclusion, SNP induced inhibition of oxidative phosphorylation, stimulation of glycolysis, but depletion of total energy production in rat reticulocytes. These alterations were accompanied with instability of energy status.     

CELL DEATH,SURVIVAL AND PROLIFERATION IN TETRAHYMENA THERMOPHILA. EFFECTS OF INSULIN,SODIUM NITROPRUSSIDE, 8-BROMO CYCLIC GMP,NG-METHYL-L-ARGININE AND METHYLENE BLUE

Cells of the ciliate Tetrahymena thermophila produce compounds that act as autocrine (paracrine) survival and/or growth factors. 8-Bromo cyclic GMP, sodium nitroprusside, hemin, protoporphyrin IX, human recombinant and bovine insulin were tested for their ability to substitute for the cell-produced factors and stimulate cell survival and proliferation. The cells were inoculated into conical flasks in a nutritionally complete, chemically defined medium at known cell densities from 5 to 5000cells/ml. In unsupplemented medium cells at 5 to 500cells/ml (‘low initial cell density cultures’) died within 8h, whereas cells at 1000 and 5000cells/ml (‘high initial cell density cultures’) proliferated with lag phases lasting for up to 4h. In the presence of insulin compounds, hemin, protoporphyrin IX, or 8-bromo cyclic GMP, cells also proliferated at all low initial cell densities. Sodium nitroprusside was effective over two separate concentration ranges: at the nanomolar levels as well at low pico- to femtomolar levels. At initial population densities of up to 50cells/ml the cells at both concentrations of sodium nitroprusside survived about 4-fold longer than the controls. At 500 initial cells/ml, cells at thehigh concentrations of sodium nitroprusside survived about 4-fold longer than those of the control cultures; they proliferated in the low concentrations of sodium nitroprusside. Concentrations of hemin, too low to have any effects on their own, had synergistic effects with sodium nitroprusside. N^G-methyl-L-arginine inhibited proliferation at high initial cell densities. This inhibitory action was reduced by high concentrations of L-arginine, protoporphyrin IX, sodium nitroprusside, or 8-bromo cGMP, but not by insulin. Methylene blue inhibited cell proliferation at high initial cell densities. This inhibition was circumvented by addition of 8-bromo cGMP. The findings that insulin-related material may be released from Tetrahymena and that insulin and sodium nitroprusside increase intracellular cGMP in these cells are discussed in relation to the presented results. Together these observations suggest that cGMP is responsible for supporting cell survival in Tetrahymena and switching the cells into their proliferative mode, and that cell-produced signal molecules and insulin stimulate an NO-dependent guanylate cyclase into producing cGMP.     

Release of tumor necrosis factor-alpha from macrophages. Enhancement and suppression are dose-dependently regulated by prostaglandin E2 and cyclic nucleotides

PGE2 has previously been shown to suppress various leukocyte functions. In this study, we examined whether PGE2 would affect release of TNF-alpha from rat resident peritoneal macrophages. Two different, dose-dependent effects were observed: low PGE2 concentrations (0.1 to 10 ng/ml) stimulated, whereas higher concentrations (greater than 10 ng/ml) suppressed TNF-alpha release. PGE2-stimulated TNF-alpha production was dependent on de novo protein synthesis and was associated with an intracellular rise of cGMP. The importance of cGMP as an intracellular messenger for PGE2 was confirmed by the following evidence: (1) low PGE2 concentrations preferentially increased cGMP and not cAMP and (2) cGMP, either exogenously added or endogenously generated by sodium nitroprusside, were efficient stimulators of TNF-alpha production. In contrast, agents increasing intracellular cAMP concentrations such as PGE1, higher PGE2 doses, isoproterenol, and theophylline, all suppressed TNF-alpha synthesis. Only resident, but not casein-elicited or Corynebacterium parvum-activated macrophages, were stimulated by low PGE2 concentrations to increase TNF-alpha production. In tumor cytotoxicity assays, PGE2-activated macrophages were active only against TNF-alpha-sensitive target cells. These findings demonstrate that TNF-alpha synthesis in macrophages is up-regulated by cGMP and down-regulated by cAMP, which indicates that cyclic nucleotides act as intracellular messengers for extracellular signals of macrophage activation.     

Concentration and regulation of cyclic nucleotides, cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells.

Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4 microM cAMP and 3.1 microM catalytic subunit of PKA, which is equivalent to 6.2 microM cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4 microM cGMP and 7.3 microM PKG monomer, equivalent to 14.6 microM cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25 microM. Treatment of washed human platelets with 10 microM (or 10 mM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27 microM (10 microM with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100 microM (or 1 microM) sodium nitroprusside elevated the platelet cGMP level to 4 microM (0.9 microM with 1 microM sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1 microM sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increase in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation.(ABSTRACT TRUNCATED AT 400 WORDS)