cGMP Radioimmunoassay Research Articles

Stimulators of the soluble guanylyl cyclase: promising functional insights from rare coding atherosclerosis-related GUCY1A3 variants.

Stimulators of the soluble guanylyl cyclase (sGC) are emerging therapeutic agents in cardiovascular diseases. Genetic alterations of the GUCY1A3 gene, which encodes the α1 subunit of the sGC, are associated with coronary artery disease. Studies investigating sGC stimulators in subjects with CAD and carrying risk-related variants in sGC are, however, lacking. Here, we functionally investigate the impact of coding GUCY1A3 variants on sGC activity and the therapeutic potential of sGC stimulators in vitro. In addition to a known loss-of-function variant, eight coding variants in GUCY1A3 were cloned and expressed in HEK 293 cells. Protein levels and dimerization capability with the β1 subunit were analysed by immunoblotting and co-immunoprecipitation, respectively. All α1 variants found in MI patients dimerized with the β1 subunit. Protein levels were reduced by 72% in one variant (p<0.01). Enzymatic activity was analysed using cGMP radioimmunoassay after stimulation with a nitric oxide (NO) donor. Five variants displayed decreased cGMP production upon NO stimulation (p<0.001). The addition of the sGC stimulator BAY 41-2272 increased cGMP formation in all of these variants (p<0.01). Except for the variant leading to decreased protein level, cGMP amounts reached the wildtype NO-induced level after addition of BAY 41-2272. In conclusion, rare coding variants in GUCY1A3 lead to reduced cGMP formation which can be rescued by a sGC stimulator in vitro. These results might therefore represent the starting point for discovery of novel treatment strategies for patients at risk with coding GUCY1A3 variants.

Relationship of Urinary cGMP Excretion with Aging and Menopausal Status in a General Population

Aging and postmenopausal women are associated with increased risks of cardiovascular disease; however, epidemiological evidence concerning the relationship of aging and the menopause with vascular biological activity is limited. We investigated the relationship of aging and the menopause with urinary excretion of cyclic guanosine 3',5' monophosphate (cGMP) in 1,541 Japanese men and women aged 40 to 79 years. The 24-hour urinary excretion of cGMP was measured with a (125)I-labeled cGMP radioimmunoassay and was adjusted for urinary creatinine excretion (nmol/mmol creatinine). Aging was positively associated with urinary excretion of cGMP for both sexes. Postmenopausal women excreted significantly less urinary cGMP than premenopausal women after adjustment for age and other cardiovascular risk factors: 48.3+/-0.04 nmol/mmol vs. 61.5+/-0.07 nmol/mmol, p=0.006. Our data suggest that cGMP-related vasodilatation is impaired in postmenopausal women.

Ginsenoside Re promotes human sperm capacitation through nitric oxide‐dependent pathway

The regulation of sperm capacitation is important for successful fertilization. Ginsenosides, the biologically effective components of ginseng, have been found to enhance intracellular nitric oxide (NO) production and the latter has recently been indicated to play a significant role in modulation of sperm functions. We investigated the effect of Ginsenoside Re on human sperm capacitation in vitro and the mechanism by which the Ginsenosides play their roles. Spermatozoa were separated by Percoll and incubated with 0, 1, 10, or 100 microM of Ginsenoside Re. The percentages of spontaneous and lysophosphatidylcholine (LPC)-induced acrosome reaction (AR), as a measure of sperm capacitation, were assayed with fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA). The intracellular cGMP level was measured by [(3)H] cGMP radioimmunoassay system. The results showed that the percentages of both spontaneous and LPC-induced AR and intracellular cGMP level were significantly enhanced by Ginsenoside Re with a concentration-dependent manner. Sodium nitroprusside (SNP, 100 nM), a NO donor, mimicked the effects of Ginsenoside Re. And pretreatment with a NOS inhibitor N(omega)-nitro-l-arginine methyl ester (L-NAME, 100 microM) or a NO scavenger N-acetyl-l-cysteine (LNAC, 1 mM) completely blocked the effects of Ginsenoside Re. Furthermore, the AR-inducing effect of Ginsenoside Re was significantly reduced in the presence of the soluble guanylate cyclase inhibitor LY83583 or cGMP-dependent protein kinase (PCK) inhibitor KT5823, whereas addition of the cGMP analogue 8-Br-cGMP significantly increased the AR of human spermatozoa. Data suggested that Ginsenoside Re is beneficial to sperm capacitation and AR, and that the effect is accomplished through NO/cGMP/PKG pathway.

Metabolic syndrome and urinary cGMP excretion in general population

To examine the relationship between metabolic syndrome and endothelial dysfunction, we investigated cross-sectionally the correlation between metabolic risk factors and urinary excretion of cyclic guanosine 3′,5′-monophosphate (cGMP), a second messenger of nitric oxide (NO), in 1541 Japanese men and women aged 40–79 years. The 24-h urinary excretion of cGMP was measured using a 125I-labeled cGMP radioimmunoassay and was adjusted for urinary creatinine excretion (nmol/mmol creatinine). The components of metabolic syndrome were defined based on the following criteria: body mass index (BMI) ≥ 25.0 kg/m 2, fasting plasma glucose ≥ 6.11 mmol/l or non-fasting plasma glucose level ≥ 11.1 mmol/l, systolic blood pressure ≥ 130 mm Hg or diastolic blood pressure ≥ 85 mm Hg, high-density lipoprotein (HDL)-cholesterol < 1.03 mmol/l for men and <1.29 mmol/l for women, and triglyceride ≥ 1.69 mmol/l. The number of components of metabolic syndrome correlated inversely with urinary cGMP excretion; means of cGMP excretion for the whole group adjusted for age, sex, and cardiovascular risk factors were 53.6, 48.6, 47.9, 44.4 and 42.3 nmol/mmol for 0, 1, 2, 3, and 4–5 components of metabolic syndrome, respectively ( p = 0.002). Our data suggest that a reduction of NO bioactivity concur with clustered features of the metabolic syndrome.

PI-25Interruption of homologous desensitization in cGMP signaling restores colon cancer cytostasis by bacterial enterotoxins

BACKGROUND Bacterial diarrheagenic heat-stable enterotoxins (STs) induce colon cancer cell cytostasis by targeting guanylyl cyclase C (GCC) signaling. Although the molecular pathway involving cGMP-dependent influx of Ca2+ through cyclic nucleotide-gated (CNG) channels has been elucidated, the long-term kinetics of the antineoplastic effects of these toxins remain undefined. METHODS GCC signaling inducing cytostasis was examined in human colon carcinoma T84 cells employing radioimmunoassay for cGMP, unidirectional 45Ca2+ current, 3H-thymidine incorporation into the DNA and flow cytometry. RESULTS Prolonged stimulation of GCC produced resistance in tumor cells to ST-induced cytostasis. Resistance reflected rapid (tachyphylaxis) and slow (bradyphylaxis) mechanisms of desensitization induced by cGMP. Tachyphylaxis was mediated by cGMP-dependent protein kinase, which reduced the influx of Ca2+ through CNG channels. In contrast, bradyphylaxis was mediated by cGMP-dependent allosteric activation of phosphodiesterase 5, which shapes the amplitude of ST-dependent cyclic nucleotide accumulation required for cytostasis. Importantly, interruption of tachy- and bradyphylaxis restored cancer cell cytostasis induced by ST. CONCLUSIONS Regimens that incorporate cytostatic bacterial enterotoxins and inhibitors of cGMP-mediated desensitization offer a previously unrecognized therapeutic paradigm for treatment and prevention of colorectal cancer. Clinical Pharmacology & Therapeutics (2005) 79, P13–P13; doi: 10.1016/j.clpt.2005.12.046

Quercetin-Induced Induction of the NO/cGMP Pathway Depends on Ca2+-Activated K+Channel-Induced Hyperpolarization-Mediated Ca2+-Entry into Cultured Human Endothelial Cells

Quercetin is one of the dietary-derived flavonoids that are held responsible for the beneficial effects of red wine drinking in coronary artery disease known as the "French paradox". We examined whether quercetin modulates endothelial function by influencing Ca2+-activated K+ channels with large conductance (BK(Ca)) in cultured human endothelial cells. Membrane potential and intracellular Ca2+ concentrations of cultured human endothelial cells derived from umbilical cord veins (HUVEC) were measured using the fluorescence dyes DiBAC, and FURA-2, respectively. NO production was examined using a cGMP radioimmunoassay. HUVEC proliferation was analyzed by cell counts and thymidine incorporation. A dose-dependent hyperpolarization of HUVEC was recorded when quercetin was added (5-100 micromol/L). The maximum effect (50 micromol/L) was significantly reduced by the addition of the highly selective BK(Ca) inhibitor iberiotoxin (100 nmol/L), but not by blockers of other Ca2+-activated K+ channels (n = 30; p < 0.05). This BK(Ca)-induced hyperpolarization caused a transmembrane Ca2+ influx, because the quercetin-induced increase of intracellular Ca2+ was blocked by iberiotoxin, or by applying 2-aminoethoxydiphenylborate (100 micromol/L)--an inhibitor of capacitative Ca2+ entry (n = 30; p < 0.05). Quercetin-induced cGMP levels were significantly reduced by the eNOS-inhibitor l-NMMA (300 micromol/L), and by iberiotoxin (n = 10; p < 0.05). Endothelial proliferation was significantly reduced by 56 % when cells were incubated with quercetin (n = 12; p < 0.05). This effect was due to the increased NO production, because it was reversed when the cells were treated with a combination of quercetin and l-NMMA. In conclusion quercetin improves endothelial dysfunction by increasing NO synthesis involving BK(Ca)-dependent membrane hyperpolarization-induced capacitative Ca 2+ entry. Increased NO production is responsible for the quercetin-dependent inhibition of endothelial proliferation.

Relationship between urinary cGMP excretion and serum total cholesterol levels in a general population

Hypercholesterolemia impairs endothelial function. However, the critical level of serum total cholesterol at which endothelial dysfunction occurs is unknown at present. We investigated cross-sectionally the correlation between urinary excretion of cyclic guanosine 3′,5′ monophosphate (cGMP), a second messenger of nitric oxide (NO) and serum total cholesterol concentrations in a general population sample of Japanese men and women. The samples comprised 1541 subjects (788 men and 753 women) aged 40–79 years, who participated in cardiovascular risk surveys between 1997 and 2002 and underwent a 24 h urine collection. Urinary excretion of cGMP was measured using a 125I-labeled cGMP radioimmunoassay and was adjusted for urinary creatinine excretion (nmol/mmol creatinine). The mean urinary cGMP excretion correlated linearly and inversely with serum total cholesterol level: mean cGMP excretion adjusted for age, sex and cardiovascular risk factors was 61.7, 53.6, 50.8, 49.2, 47.3 and 46.4 nmol/mmol for total cholesterol levels <4.14, 4.14–4.64, 4.65–5.16, 5.17–5.68, 5.69–6.20 and ≥6.21 mmol/L, respectively ( p = 0.007). This relation was more evident among individuals with end-organ damage, among subjects with higher C-reactive protein (CRP) levels and among postmenopausal women. Our data suggest a reduction of NO bioactivity with higher serum total cholesterol levels, even within clinically normal cholesterol levels.

Basic fibroblast growth factor-induced endothelial proliferation and NO synthesis involves inward rectifier K+ current.

Inward rectifier K+ currents (K(ir)) determine the resting membrane potential and thereby modulate essential Ca2+-dependent pathways, like cell growth and synthesis of vasoactive agents in endothelial cells. Basic fibroblast growth factor (bFGF) acts as a vasodilatator and angiogenic factor. Therefore, we investigated the effect of bFGF on K(ir) and assessed the role in proliferation and nitric oxide (NO) formation of endothelial cells. Using the patch-clamp technique, we found characteristic K(ir) in human umbilical cord vein endothelial cells (HUVEC), which were dose-dependently blocked by barium (10 to 100 micromol/L). Perfusion with bFGF (50 ng/mL) caused a significant increase of K(ir), which was blocked by 100 micromol/L barium (n=18, P<0.01). The bFGF-induced HUVEC proliferation was significantly inhibited when using 50 to 100 micromol/L barium (n=6; P<0.01). NO production was examined using a cGMP radioimmunoassay. bFGF caused a significant increase of cGMP levels (n=10; P<0.05), which were blocked by barium. Modulation of K(ir) plays an important role in bFGF-mediated endothelial cell growth and NO formation.

Lysophosphatidylcholine-induced modulation of Ca 2+-activated K +channels contributes to ROS-dependent proliferation of cultured human endothelial cells

Proliferation of endothelial cells plays a crucial role in the process of atherosclerotic plaque destabilization. The major component of oxidized low-density lipoprotein lysophosphatidylcholine (LPC) has been shown to promote endothelial proliferation by increasing the production of reactive oxygen species (ROS). Since K + channels are known to control the cell cycle, we investigated the role of Ca 2+-activated K + channels (BK Ca) in the regulation of LPC-induced endothelial proliferation and ROS generation. A significant increase of cell growth induced by LPC (20 μmol/l; cell counts (CCs): +87%, thymidin incorporation: +89%; n = 12, P < 0.01) was observed, which was inhibited by the BK Ca inhibitor iberiotoxin (IBX; 100 nmol/l), by the NAD(P)H-oxidase inhibitor diphenyleneiodonium (5 μmol/l) and by transfection with antisense (AS) oligonucleotides against NAD(P)H oxidase, whereas N G-monomethyl- l-arginine ( l-NMMA) further increased LPC-induced cell growth. Using the patch-clamp technique a significant increase of BK Ca open-state probability (control: 0.004 ± 0.002; LPC: 0.104 ± 0.035; n = 21, P < 0.05) by LPC was observed. Using dichlorofluorescein fluorescence microscopy a significant increase of ROS induced by LPC was reported, that was blocked by IBX and Ca 2+ antagonists. Intracellular Ca 2+ measurements revealed a capacitative Ca 2+ influx caused by LPC. Bioactivity of nitric oxide (NO) was measured using a [ 3H]-cGMP radioimmunoassay. LPC significantly decreased acetylcholine-induced NO synthesis. LPC significantly increased cGMP levels in endothelial cells transfected with AS, which was blocked by IBX. In conclusion, our results demonstrate that LPC activates BK Ca thereby increasing ROS production which induces endothelial proliferation. In addition LPC-induced BK Ca-activation contributes to increased cGMP levels, if ROS production is prevented by AS.

Chronic hypoxia attenuates cGMP-dependent pulmonary vasodilation.

Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3'5'-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.

Nitric oxide synthase activity in the dorsal periaqueductal gray of rats expressing innate fear responses.

Immunohistochemical studies have shown nitric oxide synthase (NOS)-positive neurons in the dorsolateral sector of periaqueductal gray (PAG), a neural site known to be critical for the expression of defensive responses. In the present study, we first characterized the dorsal PAG (dPAG) NOS, and then examined NOS activity and cyclic GMP (cGMP) accumulation in the dPAG of rats exposed to a predator (cat) for 15 min. NOS activity evaluated by enzymatic conversion of [3H]arginine to [3H]citrulline in dPAG of exposed rats increased 14.6% and the cGMP radioimmunoassay showed an increase of 30.6% in relation to non-exposed rats. These results suggest an involvement of the NO/cGMP pathway in the dPAG during defensive responses.

Expression and function of recombinant endothelial nitric oxide synthase gene in canine basilar artery.

Endothelial NO synthase (eNOS) is an enzyme responsible for the production of a potent vasodilator and a key regulator of vascular tone, NO. In peripheral arteries, expression of a recombinant eNOS gene increases production of NO in the blood vessel wall. This approach appears to be a promising strategy for gene therapy of cerebrovascular disease. The major objective of the present study was to determine whether a recombinant eNOS gene (AdCMVNOS) can be functionally expressed in cerebral arteries. Replication-defective recombinant adenovirus vectors encoding bovine eNOS and Escherichia coli beta-galactosidase (AdCMVLacZ) genes, driven by the cytomegalovirus promoter, were used for ex vivo gene transfer. Rings of canine basilar artery were incubated with increasing titers of the vectors in MEM. Twenty-four or forty-eight hours after gene transfer, expression and function of AdCMVNOS were evaluated by (1) immunohistochemical staining, (2) isometric tension recording, and (3) cGMP radioimmunoassay. Transfection with AdCMVNOS resulted in the expression of recombinant eNOS protein in the vascular adventitia and endothelium, associated with significantly reduced contractile responses to UTP and enhanced endothelium-dependent relaxation to calcium ionophore A23187. Basal production of cGMP was significantly increased in the transfected vessels. The reduced contractions to UTP with increased cGMP production were reversed by a NOS inhibitor, N(G)-monomethyl-L-arginine. Contractions to UTP or production of cGMP were not affected in arteries transfected with AdCMVLacZ reporter gene. The results of the present study represent the first successful transfer and functional expression of recombinant eNOS gene in cerebral arteries. Our findings suggest that cerebral arterial tone can be modulated by recombinant eNOS expression in the vessel wall.

Human endothelial cells bioactivate organic nitrates to nitric oxide: implications for the reinforcement of endothelial defence mechanisms

Although in therapeutic use for more than a century, the mode of cellular action of organic nitrates remains incompletely understood. Despite ample experimental evidence from animal studies to show that nitrates are metabolized to NO in the vascular smooth muscle, direct demonstration of such an activity in human vascular cells is still lacking. Moreover, the role of the endothelium in modulating the pharmacodynamic action of nitrates is far from clear. We therefore aimed to investigate whether or not human endothelial cells are capable of bioactivating these drugs to NO and whether the amounts generated are sufficient to elicit any biological effects. Using cultured human umbilical vein endothelial cells (HUVECs) as an established model system a combination of three different methods was used to address this issue: (1) quantification of NO formation upon endothelial nitrate metabolism using the oxyhaemoglobin technique; (2) evaluation of the second messenger response using radioimmunoassay for cGMP; and (3) assessment of mechanism and extent of potentiation of the anti-aggregatory effect of nitrates in the presence of endothelial cells as a relevant bioassay. We now show that superfusion of cultured human endothelial cells on microcarrier beads with either glyceryl trinitrate (GTN) or isosorbide dinitrate (ISDN; both at 0.1-100 mumol L-1) results in a concentration-dependent formation of NO. NO generation from isosorbide 5-mononitrate (IS-5-N) was below the detection limit. The amounts of NO produced (maximally 2.97 +/- 0.98 pmoles NO min-1 x mg protein with 100 mumol L-1 GTN; n = 8) were similar to those elicited upon challenge of the cells with 100 nM bradykinin. NO formation from either organic nitrate was accompanied, in a concentration-dependent and methylene blue-inhibitable manner, by stimulation of endothelial soluble guanylyl cyclase with consequent increases in the intracellular level of cGMP (maximally 32-fold over basal levels with ISDN), a significant portion of which was released into the extracellular space. Upon continuous 30 min superfusion or repeated application of high concentrations of GTN (100 mumol L-1) nitrate bioactivation to NO was subject to partial tachyphylaxis. Co-incubation of washed human platelets with HUVECs potentiated the anti-aggregatory action of nitrates in a cell number dependent and oxyhaemoglobin-sensitive manner and this effect, too, was accompanied by increases in intraplatelet cGMP levels. The potentiating effect was largely inhibited after blockade of sulfhydryl groups by pre-incubation of HUVECs with N-ethylmaleimide and completely abrogated after pretreatment of cells with the tissue fixative glutaraldehyde. These results demonstrate that human endothelial cells are capable of bioactivating organic nitrates to NO by an enzymatic, apparently thiol-sensitive pathway, in quantities sufficient to influence endothelial and platelet function. Besides the well known vasorelaxant action of organic nitrates, which is mainly due to their metabolism in the smooth muscle compartment, these drugs may therefore be endowed with a hitherto underestimated potential to directly influence endothelial functions via the NO/cGMP pathway. Through specific bioactivation in the endothelium itself organic nitrates can thus mimic and reinforce protective functions normally served by a functional endothelium such as the modulation of blood cell/vessel wall interactions and inhibition of cell proliferation.

Methodological variables during analysis of in vivo cerebellar cyclic GMP, an indirect marker of nitric oxide release

In light of the recent recognition of the physiological significance of nitric oxide, there is considerable interest in the methodological variables that can confound the results of the cerebellar cGMP analysis from in vivo experiments. In this study, using male Swiss Webster mice, the effect of such methodological variables as 1) weight of the animals; 2) tissue extraction procedures used in radioimmunoassay for cGMP; and 3) the commercial source of the assay kit on, harmaline-, pentylenetetrazole- or SNAP-induced increase in cerebellar cGMP in vivo were evaluated. Results indicate that mice in the 15- to 19-g weight range are most sensitive and best suited for in vivo drug effects on cerebellar cGMP. Furthermore, for the extraction of cerebellar cGMP, use of ice-cold 0.5 N hydrochloric acid and subsequent dilution of the sample in assay buffer is the simplest and fastest method. Present data also indicate that the source of the radioimmunoassay kit has a significant effect on the cerebellar cGMP results. Based on the present results, the protocol developed and the guidelines drawn are timely and of high practical significance for research in the area of pharmacology of nitric oxide.

A simple and sensitive bioassay method for detection of EDRF with RFL-6 rat lung fibroblasts.

Detailed characteristics of a new bioassay method for detection and quantification of endothelium-derived relaxing factor (EDRF) are described. Guanosine 3',5'-cyclic monophosphate (cGMP) responses of RFL-6 rat fetal lung fibroblast cells were utilized to estimate the activity of nitric oxide (NO) and EDRF. The conditioned medium from bovine aortic endothelial (BAE) cells cultured in tissue culture plates was quickly transferred to RFL-6 incubations to determine EDRF. In the presence of superoxide dismutase, RFL-6 cells cultured in six-well tissue culture plates exhibited very high sensitivities to both NO and EDRF; e.g., they responded to NO at a concentration as low as 2 nM and the basal release of EDRF from 1-2 X 10(6) BAE cells. Based on the lower detection limit of the radioimmunoassay for cGMP, calculations reveal that 100-200 fmol of NO and the basal EDRF release from 1-2 X 10(5) BAE cells can be detected with RFL-6 cells by choosing smaller culture wells. Thus this method is more sensitive than any other currently available. Furthermore, it may be widely used, since the instrumentation required is presently available in many laboratories. This bioassay technique for EDRF and NO is sensitive, simple, and quite useful for the evaluation of experimental conditions and compounds that regulate EDRF release from various endothelial cells and tissues.

The differentiation of human gastric adenocarcinoma cell line MGc80-3 induced by dibutyryl cAMPin vitro

For providing some experimental basis in establishing malignant phenotypic reversed indexes of gastric carcinoma cells, human gastric adenocarcinoma cell line MGc-80-3 was induced by dBcAMP in vitro to appraise the effect of gastric carcinoma cell differentiation by chemical inducers. Under light microscope, MGc 80-3 cells, after treated with 1 mM dBcAMP, tended to be flat and disperse, and their volume gradually enlarged, with their nucleus relatively smaller and their shape rather regular. Morphological changes, became like normal differentiated epithelial cells, were observed. The cells attached firmly, grew slowly, their growth curve showed inhibitory rate amounted to 52.87%, and cellular division exponent displayed their peak value 1.5 times less than that of MGc 80-3 cells. It was clear that dBcAMP could effectively inhibit the multiplication activity of MGc-80-3 cells. In the cells after dBcAMP treatment, remarkable changes of cell surface charges was indicated by cell electrophoresis, the ratio dropped to 3.043 from 3.988, and their retardant ratio reached up to 31.2%. cAMP content in cells after this treatment, detected by cAMP and cGMP radioimmunoassay, was enhanced by 2.42 times; and cAMP/cGMP ratio, by 1.73 times. Thus, cAMP level within MGc 80-3 cells was raised obviously by dBcAMP. Heterotransplantation experiments showed that tumoriferous rate of MGc 80-3 cells (transplanted subcutaneously to BABL/c mice) amounted to 100%, and that of the cells after this treatment was only 5.6%. Their tumorigenic ability was extremely reduced. These results fully confirmed that dBcAMP was able to change MGc 80-3 cell's malignant phenotypic characteristics and produce a reversed alteration; thus, it has a remarkable inductive effect in differentiating gastric carcinoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)

ISLET CONTENTS OF CYCLIC 3′,5′-GUANOSINE MONOPHOSPHATE UNDER CONDITIONS WHICH AFFECT THE CYCLIC 3′,5′-ADENOSINE MONOPHOSPHATE

The sensitivity of the radioimmunoassay for cGMP was considerably increased by previous 2'-O-succinylation of the nucleotide. The basal content of cGMP in beta-cell-rich pancreatic islets isolated from ob/ob-mice was similar to that of cAMP, i.e. about 3 mumoles per kg dry weight. Extra-cellular Ca2+ was a prerequisite for maintaining this amount of cGMP. The islet cGMP differed from cAMP in being only slightly enhanced or not affected at all when the islets were exposed to high concentrations of glucose, the sulphydryl reagents chloromercuribenzene-p-sulphonic acid and iodoacetamide, or the potent phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine. The data obtained suggest that the turnover rate for cGMP is much slower than that for cAMP in the pancreatic beta-cells. The interrelationships between the two cyclic nucleotides do not seem to fit into a simple pattern of antagonism.