Human Leukocyte Cyclic AMP and Cyclic GMP Levels during Chemotaxis in Delayed Type Hypersensitivity

Ten nickel-allergy patients and six healthy control subjects participated in a study of the morphology kinetics and evolution of the AMP and GMP concentration of migrated leukocytes, using an improved skin chambet technique. Also studied was the effect of nickel exposure in the patients Nickel exposure had a specific effect on the morphology from the 24th hour to the end of the 48 h observation period, with a significant increase in the percentages of basophils, eosinophils and lymphocytes and a decrease of neutrophils. A Significantly increased leukocyte migration rate (LMR) was observed from the 27th to 39th hour in six of the allergic patients exposed to nickel. There were no specific permanent changes in cAMP and cGMP concentration during nickel exposure. The control chambers of the allergy patients and health control had identical leukocyte morphology, LMR and leukocyte concentrations of cAMP and cGMP. However no correlations were found between LMR cAMP and cGMP in the eczema patients throughout the observation period.     

Cyclic AMP and cyclic GMP phosphodiesterase activities in Hodgkin's disease lymphocytes

Cyclic nucleotide phosphodiesterase (PDE) activities were studied in peripheral blood monocyte-depleted lymphocytes and enriched T-lymphocyte suspensions from thirteen patients with previously untreated Hodgkin's disease (HD) and fourteen age and sex matched healthy volunteers. Monocyte-depleted lymphocytes from HD patients showed PDE-activities which were two times higher than in their normal counterpart cells. The mean cAMP-PDE activity present in enriched HD T-lymphocyte suspensions was four times higher than in control T-lymphocytes, and the mean cGMP-PDE associated with HD T-lymphocytes was three times higher than in the controls. The hydrolytic activities present in both monocyte-depleted and T-lymphocyte enriched cells suspensions remained unchanged in absence or in the presence of calmodulin and calcium. Since depressed cAMP and cGMP resting levels have been observed in HD lymphocytes and lymphocyte subpopulations, our results suggest that the elevated PDE activities are, at least in part, responsible for the alterations in lymphocyte cyclic nucleotide levels.     

Microassay of cyclic nucleotides in vessel wall: V. Simultaneous assay of cyclic AMP and cyclic GMP

Cyclic GMP (cGMP) is an important key substance in cell function; however, the related mechanisms have not been entirely elucidated, as the content of this nucleotide within the cell is so small that an accurate estimation is difficult. We designed a microassay for cyclic GMP in 500 μg dry weight of tissue samples, using succinylation, and were able to assay, separately, the content of cyclic GMP in the intima or media of the arterial wall. In addition, we attempted to develop a method for a simultaneous determination of both cyclic AMP and cyclic GMP in a small amount of the same assay mixture. Five hundred micrograms dry weight of intima or media of rabbit aorta was prepared under a stereomicroscope. After deproteinization and lyophilization, the samples were dissolved with 60 μl of H₂O and 30 μl of succinylation mixture. After succinylation, 40 μl of each of the tissue extractions was sampled to determine the levels of cyclic AMP and cyclic GMP. The radioimmunoassay was performed, using Yamasa's kit. The recovery rates of cyclic AMP and cyclic GMP in our method were 86.2 ± 1.2 and 87.6 ± 1.3%, respectively. The levels of cyclic GMP in the intima and media of aorta of cow, pig, and rabbits were estimated to be, intima: 0.18 ± 0.02, 0.06 ± 0.03, and 0.20 ± 0.01 pmole/mg protein, media: 0.08 ± 0.01, 0.04 ± 0.01, and 0.09 ± 0.01. The cyclic GMP levels in the intima of aorta of cow and rabbits were high compared to levels in the media and this difference was statistically significant (P < 0.05).     

Alterations of peripheral blood lymphocyte cyclic AMP and cyclic GMP in untreated patients with hodgkin's disease

Cyclic AMP and cyclic GMP are important regulatory agents of lymphocyte functions. Depressed T-lymphocyte functions are frequently associated with Hodgkin's disease and suppressor monocytes have been implicated in the pathogenesis of this defect. In the present study cAMP and cGMP resting levels were measured in lymphocytes from 18 untreated patients with Hodgkin's disease using a sensitive radioimmunoassay. A significant decrease of cAMP (P less than 0.001) and, to a lesser degree, of cGMP (P less than 0.01) was found in monocyte-depleted lymphocyte suspensions from the patients compared to controls. Studies of patient and control lymphocyte subpopulations showed in patients a clear deficit of cAMP in T-depleted lymphocytes, rather than in T cells, with a low cAMP/cGMP molar ratio in both subpopulations. From this data it is clear that factors other than prostaglandin-mediated suppression of monocyte origin are involved in the pathogenesis of the T-lymphocyte depression associated with Hodgkin's disease.     

Atrial natriuretic factor increases cyclic GMP and cyclic AMP levels in a directly photosensitive pineal organ

Atrial natriuretic factor (ANF) stimulates accumulation of cyclic GMP in a photosensitive organ, as evidenced for the first time in cultured trout pineals. Stimulation was rapid (within a few min), dose-dependent, and stronger in organs cultured in darkness than in those cultured under light. After 30 min in the dark, (i) cyclic AMP levels were slightly increased at 10^–7 mole/l of ANF, (ii) cyclic GMP and cyclic AMP increased dramatically after inhibition of the phosphodiesterases by isobu-tylmethylxanthine (IBMX), (iii) ANF and IBMX effects were more than additive on cyclic GMP, (iv) pertussis toxin decreased the cyclic GMP response to ANF. These responses were affected by light. The possibility that cyclic GMP might be a second messenger of both light and chemical (ANF) inputs, in pineal photoreceptor cells, is hypothetized.     

Oxytocin affects the release of steroids, insulin-like growth factor-I, prostaglandin F2{alpha} and cyclic nucleotides by human granulosa cells in vitro

The aim of the present experiments was to examine the effectsof oxytocin (1–10 000 ng/ml) on hormone and cyclic nucleotidesecretion by human granulosa cells cultured in a serum-supplementedmedium. The release of progesterone, oestradiol, insulin-likegrowth factor-I, prostaglandin F2 cAMP and cGMP into the incubationmedium was analysed by radioimmunoassay. An inhibition of progesterone,but not of oestradiol release was observed. Oxytocin also stimulatedinsulin-like growth factor-I, prostaglandin F2 cAMP and cGMPoutput The results suggest an involvement of oxytocin in theautocrine/paracrine regulation of steroid, insulin-like growthfactor, prostaglandin and cyclic nucleotide release by humanovarian cells.     

Pyrophosphate-induced inflammation: An in vivo study of the interrelationship of intracellular cyclic AMP and cyclic GMP

The changes in leucocyte concentrations of cyclic AMP and cyclic GMP were monitored during pyrophosphate-induced pleurisy in rats. A classical Yin Yang relationship was demonstrated.     

The relationship between human sperm apoptosis, morphology and the sperm deformity index

BACKGROUND: This study aimed to assess the relationship between apoptosis in human ejaculated spermatozoa, sperm morphology and the novel sperm deformity index (SDI). METHODS: Semen specimens from 50 healthy donors were prepared by density-gradient centrifugation followed by incubating the prepared sperm with paramagnetic annexin V-conjugated microbeads and subjecting this to magnetic cell sorting (MACS). The procedure delivers two sperm fractions: annexin-negative (non-apoptotic) and annexin-positive (apoptotic). Activated caspase-3 levels and the integrity of the sperm mitochondrial membrane potential (MMP) were assessed as markers of apoptosis in the annexin-negative and -positive aliquots following MACS. Sperm morphology and the SDI scores were assessed using the strict criteria. RESULTS: Compared with the apoptotic sperm subpopulations, the non-apoptotic sperm subpopulations had an improved sperm morphology profile as demonstrated by significantly higher proportions of sperm with normal morphology and significantly lower SDI scores and percentages of sperm with acrosomal defects, midpiece defects, cytoplasmic droplet and tail defects. There was a significant correlation between sperm morphology attributes studied and the expressed apoptotic markers - caspase-3 activation and MMP integrity. CONCLUSIONS: Non-apoptotic sperm fractions have morphologically superior quality sperm compared with apoptotic fractions as reflected by significantly lower SDI scores. The study results may support abortive apoptosis, where the apoptotic mechanism of sperm is already triggered prior to ejaculation.     

GTP-related difference in cyclic AMP production between resident and inflammatory human peritoneal macrophages.

In macrophages cyclic AMP (c-AMP) plays an important role in regulating many activities such as phagocytosis, migration and tumoricidal activity. High intracellular levels of c-AMP are negatively correlated with these activities. In earlier studies we have shown that c-AMP levels in inflammatory human peritoneal macrophages (IM) were markedly lower when compared to levels in resident macrophages (RM). This is in line with the fact that c-AMP down-regulates macrophage activity. To our knowledge no data are available on the mechanism underlying the difference in c-AMP production between RM and IM. In this study the difference in c-AMP production between RM and IM has been investigated on the level of receptor and G-protein-related mechanisms. Macrophage membranes were incubated with different agents i.e. prostaglandin E2 (PGE2), prostacyclin I2 (PGI2), isoprenalin (ISO) and sodium fluoride (SF). Additionally, the capacity of IM and RM to hydrolyse quanosine triphosphate (GTP) was measured. Only in the presence of GTP (10(-4) M) could the c-AMP difference be detected (RM = 51 +/- 4.4 pmol/mg protein/min +/- S.E., n = 22, IM = 32.8 +/- 5.2 pmol/mg protein/min +/- S.E., n = 10, p less than 0.01). After receptor stimulation with PGE2, PGI2 and ISO, c-AMP levels increased to the same extent in both IM and RM with no effect on the GTP-related difference. After SF stimulation, c-AMP levels in RM and IM increased to the same level (RM = 63 +/- 8 pmol/mg protein/min, n = 14, IM = 58 +/- 11 pmol/mg protein/min, n = 13).(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between human sperm acrosin, acrosomes, morphology and fertilization in vitro

Acrosin was measured in the semen used for sperm preparation for in-vitro fertilization (IVF) in 118 patients. Acrosin levels correlated with the proportion of spermatozoa with normal intact acrosomes determined with Pisum sativum agglutinin labelled with fluorescein. However, acrosin levels and the proportion of spermatozoa with normal intact acrosomes in semen were not significantly related to the fertilization rate in vitro. Only the percentage normal morphology and sperm concentration in the insemination medium were independently significantly related to the fertilization rate by logistic regression analysis. In patients with fewer than 30% of spermatozoa with normal morphology, although acrosin levels were not correlated with the fertilization rate, the proportion of spermatozoa with normal intact acrosomes in the insemination medium was the only significant factor in the logistic regression analysis. In conclusion, acrosin levels have no prognostic value for fertilization in vitro but the proportion of spermatozoa with normal intact acrosomes may be a useful clinical marker of fertilizing ability in men with poor sperm morphology.     

Effect of taurine on the cyclic AMP and GMP levels in the rat heart during stress

Taurine had no effect on the cyclic nucleotide level in the heart of intact rats but sharply reduced the increase in cyclic AMP and cyclic GMP levels taking place during stress. The action of taurine on the cyclic GMP content in the heart was not exhibited after preliminary atropinization of the animals; its effect on cyclic AMP was greatly reduced after partial blockade of -adrenoreceptors. It is suggested that taurine is a nonspecific regulator of the sensitivity of the myocardial cells to biologically active substances.All-Union Cardiologic Scientific Center, Academy of Medical Sciences of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR, E. I. Chazov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 2, pp. 134–137, February, 1979.     

Negative inotropic effect of cyclic GMP in cardiac fiber fragments

Summary The force of spontaneously beating cardiac cellular fragments obtained from mice heart by homogenization was recorded in presence of cyclic guanosine –3.5-monophosphate (cGMP) and cyclic 8-bromguanosine –3.5-monophosphate in concentrations of 3×10^–6 M –33×10^–6 M. The nucleotide decreased the force and reduced the rate of spontaneity. Eventually the preparation became quiescent. It is thought that this nucleotide either reduces the capacity to sequester calcium or affects its release from the sarcotubular system.     

低水平辐射对大鼠血浆和某些腺体中cAMP和cGMP水平的影响

本研究分二批进行,第一批用成年雄性大鼠每天接受X射线全身照射50mGy/次和100mGy/次,剂量率为15mGy/分,6次/周,共照5周(30次),累积剂量分别为1.5Gy和3.0Gy。受照大鼠睾丸重量在停照后4周内呈持续性减轻,以照射剂量大,停照时间长者,减轻最明显(相当于对照43.9%)。睾丸中cAMP含量于停照后1周升高,以100mGy组明显(P<0.01),停照后2周仍显著高于对照(P<0.05),停照后4周回降到正常较低水平。而血浆和肾上腺中cAMP和胸上腺中cGMP含量无明显变化。第二批实验动物条件与第一批相同,每天接受16.3mGy,~(60)Coγ射线全身照射,剂量率为45.3pGy/分,6次/周,每次照射6小时,在照射6、10、14、18、22周,累积剂量分别为0.59、0.98、1.37、1.76、2.15Gy时与相应对照组比较,垂体、睾丸中cAMP含量及睾丸重量无显著差别,证实慢性小剂量全身照射对睾丸组织的损伤主要取决于剂量率大小,剂量率愈大损伤愈明显。     

Interleukin-1 alpha modulates luteinizing hormone stimulated cyclic AMP and progesterone release from human granulosa cells in vitro.

This study examines the possible direct effect of interleukin-1 alpha (IL-1 alpha) upon human granulosa cells. The cells were isolated from follicles of stimulated cycles in women undergoing oocyte retrieval for in-vitro fertilization. Purified cell preparations were cultured for different time periods in the presence of IL-1 alpha and human luteinizing hormone (LH) or follicle stimulating hormone. IL-1 alpha stimulated basal as well as LH-induced progesterone accumulation. The response in terms of cyclic AMP was more complex, there was no effect of IL-1 alpha on basal cyclic AMP accumulation. However, at the highest concentration tested (50 IU/ml), IL-1 alpha enhanced cyclic AMP accumulation over that seen with LH alone. At a lower concentration, IL-1 alpha either had no effect or was slightly inhibitory to the LH-induced cyclic AMP accumulation, depending on the culture period. Our results, taken together with other findings, are compatible with the view that IL-1 alpha has a potential regulatory role in the granulosa-luteal cell transition in the human ovary.     

Stimulus-secretion coupling: role of cyclic AMP, cyclic GMP and calcium in mediating enzyme (kallikrein) secretion in the submandibular gland.

1. The role of adenosine 3':5'-phosphate (cyclic AMP) and guanosine 3':5'-phosphate (cyclic GMP) as second messengers for the enzyme secretory response evoked by the autonomic neurotransmitters, noradrenaline and acetylcholine, is examined in this in vitro study on the guinea-pig submandibular gland. 2. Noradrenaline increased enzyme (kallikrein) secretion. The initial stimulation of enzyme release appeared to be dose-dependent. The time course of cumulative kallikrein secretion revealed a complex pattern. Isoprenaline and phenylephrine were almost as potent as noradrenaline in releasing kallikrein. Both propranolol and phentolamine were required to fully inhibit the noradrenaline-stimulated enzyme secretion. 3. The cumulative secretion of kallikrein evoked by acetylcholine was dose-dependent. The onset of secretion showed a significantly greater time-lag than that observed with noradrenaline. Atropine effectively blocked the release of kallikrein by acetylcholine. 4. Dibutyryl cyclic AMP stimulated enzyme secretion. Dibutyryl cyclic GMP caused an initial increase which was not maintained. 5. The cyclic nucleotide phosphodiesterase inhibitors, theophylline and papaverine, increased basal kallikrein secretion. The action of the cyclic phosphodiesterase inhibitors on the secretory response to noradrenaline, acetylcholine, dibutyryl cyclic AMP and dibutyryl cyclic GMP was complex. In general, the increase in enzyme release produced by the secretagogues was additively enhanced by both inhibitors. 6. Omission of calcium inhibited both acetylcholine and dibutyryl cyclic GMP stimulated kallikrein release, but to a lesser degree than that of noradrenaline and dibutyryl cyclic AMP. High concentrations of extracellular calcium (10 mM) appeared to enhance the action of acetylcholine. 7. Noradrenaline produced a rise in the intracellular level of cyclic AMP. The increase preceded the stimulated secretion of kallikrein. Of the various adrenergic agonists, noradrenaline and isoprenaline were the most potent, whereas phenylephrine was significantly less effective in raising basal cyclic AMP values. Acetylcholine was without effect, even in the presence of a cyclic phosphodiesterase inhibitor. 8. Acetylcholine and noradrenaline raised intracellular levels of cyclic GMP only when the tissue incubations were performed in the presence of a cyclic phosphodiesterase inhibitor. The increase in cyclic GMP produced by acetylcholine preceded enzyme secretion. 9. Morphological data substantiated the finding that the in vitro release of kallikrein evoked by the secretagogues was associated with the depletion of secretory granules and vacuolations in acinar cells of the gland slices. 10. The molecular mechanisms which control enzyme secretion in the exocrine submandibular gland are discussed. Models are presented for the role of transmitter-specific cyclic nucleotides and calcium in stimulus-secretion coupling.     

Relationship between systemic nitric oxide metabolites and cyclic GMP in healthy male volunteers

Aim: Nitric oxide (NO) is an endogenous mediator of many physiological processes, many of which are mediated by cyclic guanosine 3′,5′‐monophosphate (cGMP). Much effort has been made to validate clinical markers of NO production or bioavailability. While the measurement of plasma nitrate, nitrite, and cGMP concentrations have been suggested to reflect endogenous production of NO, there is no study showing whether there is correlation between these three markers. In the present study, we investigate whether there is correlation between the plasma concentrations of nitrate, nitrite, and cGMP in a relatively homogeneous group of 141 healthy subjects. Methods: Venous blood samples were collected from healthy male subjects and plasma aliquots were then immediately removed and stored at ?70 °C until analysed in duplicate for their nitrite and nitrate content using ozone‐based chemiluminescence assays. Plasma cGMP levels were determined by using a commercial enzyme immunoassay. Results: While we found no significant correlation between plasma nitrite and nitrate concentrations (P = 0.747), or between plasma nitrate and cGMP concentrations (P = 0.221), a significant positive correlation was found between plasma cGMP and nitrite concentrations (P = 0.017, r_s = 0.270). Conclusions: The significant correlation we found between plasma nitrite and cGMP concentrations is consistent with the notion that nitrite or cGMP concentrations in plasma may be useful clinical markers of NO formation in healthy subjects.     

Comparative genomic hybridization analysis of human oocytes and polar bodies

BACKGROUND: Classical cytogenetic methods and fluorescent in situ hybridization (FISH) have been employed for the analysis of chromosomal abnormalities in human oocytes. However, these methods are limited by the need to spread the sample on a microscope slide, a process that risks artefactual chromosome loss. Comparative genomic hybridization (CGH) is a DNA-based method that enables the investigation of the entire chromosome complement. We optimized and evaluated a CGH protocol for the chromosomal analysis of first polar bodies (PBs) and oocytes. The protocol was then employed to obtain a detailed picture of meiosis I errors in human oogenesis. METHODS: 107 MII oocyte-PB complexes were examined using whole genome amplification (WGA) and CGH. RESULTS: Data was obtained for 100 complexes, donated from 46 patients of average age 32.5 (range 18-42). 22 complexes from 15 patients were abnormal, giving an aneuploidy rate of 22%. CONCLUSIONS: The results presented in this study more than double the quantity of CGH data from female gametes currently available. Abnormalities caused by whole chromosome non-disjunction, unbalanced chromatid predivision and chromosome breakage were reliably identified using the CGH protocol. Analysis of the data revealed a preferential participation of chromosome X and the smaller autosomes in aneuploidy and provided further evidence for the existence of age-independent factors in female aneuploidy.