Levels Of Cyclic Adenosine Research Articles

Dihydroartemisinin treatment exhibits antitumor effects in glioma cells through induction of apoptosis.

The present study aimed to investigate the effect of dihydroartemisinin on the proliferation of chemotherapy‑resistant C6 rat glioma cells. The results revealed that incubation of C6 glioma cells with a range of dihydroartemisinin concentrations for 48h led to a significant (P<0.02) reduction in the cell number. There was a ‑0.8-fold reduction in the cell count following treatment with 20µM dihydroartemisinin when compared with the control cultures. Analysis of DNA synthesis using bromodeoxyuridine (BrdU) staining demonstrated a reduction in the BrdU‑labeling index (LI) following treatment with 20µM dihydroartemisinin. There was a 6‑fold reduction in the BrdU‑LI compared with the control cultures. Incubation of the C6glioma cells with dihydroartemisinin led to a concentration dependent reduction in the level of cyclic adenosine 3',5'‑monophosphate following 48h. The percentage of apoptotic cells in the cultures incubated with 20µM dihydroartemisinin was 54.78% compared with 2.57% in the control cultures. Incubation of the C6 glioma cells with dihydroartemisinin for 48h led to a reduction in the percentage of cells in G2/Mphase with an increase in G0/G1phase. The control cells exhibited spindle‑shaped morphology and were actively undergoing mitosis following 48h of culture. The morphological characteristics of the cells treated with dihydroartemisinin were demonstrated to be round with small surface projections. Therefore, treatment of glioma cells with dihydroartemisinin exhibited an antitumor effect by the induction of apoptosis. Therefore, dihydroartemisinin should be evaluated further in the animal models for the treatment of glioma.

Brain-Derived Neurotrophic Factor Stimulates Production of Prostacyclin in Cerebral Arteries

The role of brain-derived neurotrophic factor (BDNF) and its receptor, tropomyosin receptor kinase B, in control of cerebral circulation is poorly understood. The present study was designed to investigate the cerebral vascular effects of BDNF in vivo. Replication incompetent adenovirus encoding either rat BDNF (AdBDNF) or green fluorescent protein was injected intracisternally into rabbits. Forty-eight hours later, animals were euthanized. Plasma and cerebrospinal fluid levels of BDNF were measured by enzyme-linked immunosorbent assay, vasomotor function of isolated basilar arteries was studied in organ chambers, protein expression in the basilar arteries was studied by Western blotting, prostanoid levels were measured by enzyme-linked immunosorbent assay, and cyclic adenosine 3',5'-monophosphate levels were measured by radioimmunoassay. The levels of BDNF in the cerebrospinal fluid were significantly elevated in AdBDNF-treated rabbits as compared with adenovirus encoding green fluorescent protein-treated rabbits (37+/-5 ng/mL versus 0.006+/-0.003 ng/mL, respectively; P<0.05; n=14). Western blotting studies revealed that in basilar arteries, AdBDNF increased protein expression of prostacyclin synthase, whereas expression of endothelial nitric oxide synthase and phosphorylated (Ser 1177) endothelial nitric oxide synthase remained unchanged. During incubation with arachidonic acid (1 micromol/L), PGI(2) production and levels of cyclic adenosine 3',5'-monophosphate were significantly elevated only in AdBDNF-treated rabbit basilar arteries (P<0.05, n=6). Relaxations to acetylcholine (10(-9) to 10(-5) mol/L) and arachidonic acid (10(-9) to 10(-5) mol/L) were significantly potentiated in basilar arteries from rabbits injected with AdBDNF. Potentiation of relaxations to acetylcholine in AdBDNF-treated basilar arteries was inhibited by the nonselective cyclooxygenase inhibitor, indomethacin (10(-5) mol/L, P<0.05, n=6) and constitutive phospholipase A(2) inhibitor, AACOCF3 (2x10(-5) mol/L, P<0.05, n=5). Our results demonstrate that in cerebral arteries, BDNF-induced activation of tropomyosin receptor kinase B receptor signaling in vivo promotes prostacyclin biosynthesis. These findings provide novel mechanistic insight into the vascular protective effect of BDNF in cerebral circulation.

Icariin attenuates chronic mild stress-induced dysregulation of the LHPA stress circuit in rats

Chronic mild stress (CMS) is suggested to develop dysregulation of the limbic-hypothalamic-pituitary-adrenal (LHPA) stress circuit. Icariin, a major constituent of flavonoids isolated from Epimedium brevicornum, has been previously confirmed to rescue the HPA axis abnormalities in animal models of depression. However, antidepressant treatment of icariin on corticotropin-releasing factor (CRF) system within the LHPA stress circuit and its interaction with serotonergic receptor are still seldom studied in CMS model of animals. The present study further investigated the effects of CMS procedure and subsequent icariin treatment on mRNA and protein levels of CRF, CRF receptor 1 (CRFR1) and CRF binding protein (CRFBP), as well as sucrose intake in rats. Moreover, the levels of cyclic adenosine 3',5'-monophosphate (cAMP) response element binding protein (CREB), glucocorticoid receptor (GR) and 5-hydroxytryptamine 1A receptor (5-HTR1A) in hypothalamus, hippocampus and frontal cortex were simultaneously evaluated for their participations in CRF system in this model. We found that CMS procedure significantly increased CRF expression levels in the brain regions, and decreased GR and 5-HTR1A in hippocampus and frontal cortex, with sucrose intake reduction representing the hedonic deficit in rats. Icariin restored these alterations in CMS rats. These results confirmed the hypothesis that icariin exerted antidepressant-like effect via its regulation of central CRF system. And hippocampus was suggested as an important neural area controlling the LHPA stress circuit in icariin-treated CMS rats. These findings for the first time proved that the potential molecular mechanism of antidepressant action of icariin was targeted on the interaction of the LHPA stress circuit and serotonergic function in CMS rats.

VP16CREB-induced differentiation of neuroblastoma

Drug-induced differentiation is commonly used as a therapeutic modality for the treatment of neuroblastoma tumors. Increased level of cyclic adenosine 3', 5'-monophosphate (cAMP) mediates terminal differentiation in some neuroblastoma cell lines through activation of several signaling networks, including cAMP response element binding protein (CREB). Objective was to test whether cAMP-induced differentiation in a murine neuroblastoma cell line (NBP2) is partly mediated by CREB. Fluorescent microscopy was used to document neuron-like morphological changes imparted by a constitutively active CREB (VP16CREB). Real time PCR (RT-PCR) was performed to verify changes in the expression of cAMP/CREB responsive genes. It was found that transient expression of VP16CREB into NBP2 cells resulted in morphological changes that were characteristics of terminally differentiated neurons. Furthermore, increased expression of cAMP responsive genes was compromised in cells resisting VP16CREB-mediated differentiation. A constitutively active CREB induces terminal differentiation in a subset of NBP2 cell population. Altered expression of cAMP responsive genes may account for differentiation resistant phenotype in NBP2 cells.

Dopamine regulates cell cycle regulatory proteins via cAMP, Ca2+/PKC, MAPKs, and NF‐κB in mouse embryonic stem cells

This study examined the effect of dopamine on DNA synthesis and its related signal cascades in mouse embryonic stem (ES) cells. Dopamine inhibited DNA synthesis in both a dose- and time-dependent manner. Dopamine, SKF 38393 (D1 receptor agonist), and quinpirole (D2 receptor agonist) decreased the level of [(3)H]-thymidine incorporation. The level of cyclic adenosine 3, 5-monophosphate (cAMP) was increased by SKF 38393 but not by quinpirole. The protein kinase C (PKC) protein was translocated from the cytosolic fraction to the membrane compartment by dopamine. Dopamine also increased [Ca(2+)](i), which was blocked by EGTA (an extracellular Ca(2+) chelator), BAPTA-AM (an intracellular Ca(2+) chelator), nifedipine (a L-type Ca(2+) channel blocker), SQ 22536 [an adenylyl cyclase (AC) inhibitor] and neomycin [a phospholipase C (PLC) inhibitor]. Dopamine, SKF 38393, and quinpirole increased the level of p44/42 mitogen-activated protein kinases (MAPKs), p38 MAPK, and stress-activated protein kinase/Jun-N-terminal kinase (SAPK/JNK) phosphorylation. Dopamine also increased level of H(2)O(2) formation and activated the transcription factor family NF-kappaB. Moreover, SKF 38393, quinpirole, and dopamine inhibited cell cycle regulatory proteins, which is consistent with the change in the level of [(3)H]-thymidine incorporation observed. The dopamine-induced decrease in cyclin E, cyclin-dependent protein kinase-2 (CDK-2), and cyclin D1, CDK-4 were blocked by pertussis toxin (G protein inhibitor), SQ 22536, neomycin, bisindolylmaleimide I (PKC inhibitor), SB 203580 (p38 MAPK inhibitor), PD 98059 (p44/42 inhibitor), and SP 600125 (SAPK/JNK inhibitor). In conclusion, dopamine inhibits DNA synthesis in mouse ES cells via the cAMP, Ca(2+)/PKC, MAPKs, and NF-kappaB signaling pathways.

Sex-differences in adrenocortical responsiveness during development in rats

It is known that the stress hyporesponsive period (SHRP), which seems to be related to an immature hypothalamo-pituitary-adrenal (HPA) regulatory system, occurs during the first 2 weeks after birth in rats. In the present study, we investigated the effects of sex-steroid hormones on adrenocortical responsiveness to adrenocorticotropic hormone (ACTH) in neonatal rats. The levels of cyclic adenosine 3′,5′-monophosphate (cAMP), corticosterone, and adenylate cyclase activity increased with the dose of ACTH in adrenal cells of males and females in vitro. The ACTH responsiveness in adrenal cells increased with age (7–35 days of age), that is, the loss in responsiveness to ACTH just after birth began to recover in 14–35-day-old rats, but the responsiveness in 14-day-old rats was attenuated in males compared with females. Although castration markedly augmented the responsiveness in male rats, testosterone-replacement in the castrated male rats inhibited the enhancement. Furthermore, the responsiveness in 14-day-intact female rats was suppressed by treatment with testosterone. Expression levels of ACTH receptor mRNA in adrenals increased with age in the female rat, but not in the male. Castration enhanced the level of ACTH receptor mRNA to three-fold of that in intact male rats at 14 days of age, but replacement treatment with testosterone in castrated male rats lowered the elevated levels. Testicular androgens are thought to evoke a gender-specific response in neonates, and the temporal decrease of adrenal ACTH-responsiveness might be due to the topically immature adrenal system as well as the central nervous system in mammals.

Quality assessment of platelet concentrates supplemented with second-messenger effectors.

While reducing the potential for bacterial contamination, the storage of platelet concentrates (PCs) at refrigerated temperatures is not routine, because of the induction of the so-called platelet storage lesion. As the modulation of second-messenger levels might help to overcome this drawback, a quality assessment of PCs treated with a mixture of second-messengers effectors known as ThromboSol was performed. The PCs were supplemented with ThromboSol or phosphate-buffered saline, and stored in parallel at 22 degrees C with continuous agitation or at 4 degrees C. At 1, 5, and 9 days, an in vitro quality assessment of the PCs was performed, including measurement of cell number, metabolic and integrity markers, platelet surface expression of glycoproteins, platelet response to ristocetin and thrombin, and levels of cyclic adenosine 3', 5' monophosphate (cAMP) and thromboxane B2 (TxB2). Control PCs stored at 4 degrees C underwent aggregation and displayed a significant decrease in the platelet number (40% on Day 5). By contrast, the ThromboSol-treated PCs maintained 80 percent of their initial platelet concentration after 9 days of storage at 4 degrees C. Compared to PCs stored at 22 degrees C, refrigerated PCs exhibited minor changes in metabolic values throughout storage, but the addition of ThromboSol induced a rise in metabolic rate during storage at 22 degrees C. Platelet responsiveness to both ristocetin and thrombin was maximally preserved in the ThromboSol-treated PCs stored at 4 degrees C. These units also maintained high levels of cAMP and low concentrations of TxB2 during storage. The pharmacologic supplementation of PCs with ThromboSol significantly favors the maintenance of in vitro integrity and responsiveness of platelets during extended storage at refrigerated temperature. This protective effect seems to be a consequence of the ability of ThromboSol's components to sustain high levels of cAMP and to inhibit TxB2 production during the entire extended-storage period.

Neurochemical changes in cerebellum of goldfish exposed to various temperatures.

Acclimation of goldfish at 35 degrees C increased the cerebellar content of aspartate, glutamate, and taurine and [3H]glutamate uptake. Acclimation at 4 degrees C increased the levels of glutamine, serine, and alanine and glutamine synthetase (GS) activity. Adenosine content increased in cerebellum of fish acclimated to warm temperature. K+-evoked release of endogenous and exogenous glutamate from cerebellar slices increased in fish acclimated at 35 degrees C compared to 4 degrees C. The basal level of cyclic adenosine 3':5'-monophosphate (cAMP) in perfused cerebellar slices in fish acclimated at 35 degrees C was much higher than in fish acclimated at 5 degrees and 22 degrees C. It is concluded that variations of environmental temperature produces large neurochemical changes in goldfish cerebellum.

Effects of lead on osteoclast-like cell formation in mouse bone marrow cell cultures.

To examine an effect of lead (Pb) on the process of osteoclast-like cell formation from its progenitors, we used a mouse bone marrow culture system in which osteoclast-like multinucleated cells (MNCs) were formed in response to bone-resorbing agents. In a 9-day culture period, Pb dose-dependently stimulated MNC formation over the concentration range 2-10 microM, whereas at 40 microM Pb, MNC formation declined. In an 11-day culture period, MNC formation reached a maximum at 5 microM Pb and decreased with increasing concentration of Pb at 10-40 microM. Pb-stimulated MNC formation was inhibited by both indomethacin and SC19220, an antagonist of prostaglandin E2 (PGE2) receptor. Pb stimulated the production of PGE2 in marrow cell cultures, suggesting that Pb-stimulated MNC formation is dependent on the production of PGE2. 3-Isobutyl-1-methylxanthine potentiated Pb-stimulated MNC formation and 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, inhibited it. A calcium ionophore A23187 increased Pb-induced MNC formation and verapamil, a calcium channel blocker, depressed it. It is possible that a PGE2-induced increase in the levels of cyclic adenosine 3',5'-monophosphate (cAMP) and calcium ions in marrow cells is involved in Pb-induced MNC formation. Pb and parathyroid hormone showed a synergistic stimulation on MNC formation. From these results, Pb is thought to induce osteoclast-like cell formation by a mechanism involving PGE2 which increases the intracellular levels of cAMP and calcium ions.

The mode of interaction of amiloride and some of its analogues with the adenosine A 1 receptor

Amiloride, a potassium sparing diuretic, inhibits adenosine A 1 receptor-radioligand binding in calf and rat brain membranes in the low micromolar range. The drug interacted with the A 1 receptor in a manner different from classical A 1 ligands, but structure-activity relationship studies indicated that this inhibitory effect is not related to the ion transport inhibiting properties of amiloride (Garritsen et al., 1990a,b) In the present study, the question is addressed how amiloride interacts with the adenosine A 1 receptor. Amiloride and two of its analogues, in concentrations equivalent to their K i values in displacement studies, decrease the affinity of the A 1 antagonist [ 3H]8-cyclopentyl-1,3-dipropylxanthine, but not the maximal binding capacity of the radioligand. Furthermore, the dissociation rate of the receptor ligand complex is unaltered in the presence of amiloride or its analogues in a concentration exceeding the K i value 10-fold. These characteristics argue for a purely competitive mode of interaction. The functional consequences of the interaction between amiloride analogues and the A 1 receptor were investigated at the level of cyclic adenosine 3′,5′-monophosphate (cAMP) formation. The amiloride analogue 5-( N-butyl- N-methyl) amiloride (MBA) reversed A 1-receptor mediated inhibition of forskolin-stimulated cAMP formation in rat fat cell membranes. In this model, the antagonist potency of MBA is ca 5 μM. This value is in fair agreement with a K i value of 3.5 μM in binding assays under similar conditions. In conclusion, amiloride inhibits A 1 receptor binding in an apparently competitive manner. This suggests that the binding sites of amiloride and the classic A 1 receptor ligands may at least partially overlap. The fact that amiloride affects adenosine A 1 receptors and a variety of other neurotransmitter receptors might be of importance for the understanding of the structure and function of receptor molecules.

α-MSH causes a small rise in cAMP but has no effect on basal or ultraviolet-stimulated melanogenesis in human melanocytes

The effects of alpha-melanocyte stimulating hormone (alpha-MSH) were studied on levels of cyclic adenosine 3',5'-monophosphate (cAMP), melanin content and response to ultraviolet radiation (UVR) in cultured human melanocytes (HuMC). Foreskin HuMC were cultured in a hormone-supplemented system not dependent on the presence of phorbol esters. Following addition of alpha-MSH (10(-6) M) there was a rise in cAMP levels maximal between 5 and 15 min to 9.4 +/- 3.2 pM/10(5) cells, while control levels were 3.6 +/- 0.7 pM/10(5) cells. After 7 days' culture in the presence of alpha-MSH (10(-8) -10(-6) M) the melanin content increased by only 35%, whereas Forskolin (10(-5) M) induced a 9.5-fold rise in cAMP after 5 min and a 10.9-fold rise in melanin content after 7 days. When HuMC were irradiated daily for 6 days with UVR (Helarium fluorescent lamps emitting 20% UVB, 80% UVA) melanin content rose 2.7-fold (SE 0.3). This was unchanged or slightly reduced in the presence of alpha-MSH (10(-8)-10(-6) M). Parallel observations on Cloudman S91 melanoma cells showed that alpha-MSH caused only an 80% increase in melanin content after 4 days. The rise in melanin content induced by three daily UV-irradiations (2.4-fold, SE 0.5) was unchanged by alpha-MSH (10(-8)-10(-6) M). Although alpha-MSH induces a small rise in cAMP in HuMC this does not result in melanogenesis, and the response to UVR is not affected by alpha-MSH in either HuMC or S91 cells.

Characterization of a monoclonal antibody which inhibits the biological activity of human chorionic gonadotrophin in human corpora lutea

In the present study a murine monoclonal antibody (MCA), directed against human chorionic gonadotrophin (HCG), was investigated in vitro utilizing human corpora lutea (CL). The CL were excised from women of fertile age undergoing various gynaecological operations. The CL specimens were cut into pieces and incubated in standard medium for 2 or 4 h in the presence of HCG, luteinizing hormone (HLH) or prostaglandin (PG) E2 alone or in combination with the MCA directed against HCG. After incubation, the tissue levels of cyclic adenosine 3',5' monophosphate (cAMP) and the media contents of progesterone (P) were determined. HCG, HLH and PGE2 stimulated both cAMP and P formation in CL of all ages, while the MCA alone had no effect on these parameters. The MCA, however, effectively counteracted the stimulatory effect of HCG on both cAMP and P formation on a 1:1 molar basis. This antagonistic effect was clearly reversible and could be overcome by increasing the HCG concentrations. The stimulatory effect of HLH and PGE2, on the other hand, was not influenced by the antibody, even when administered at high concentrations. These in-vitro data show that the MCA studied effectively counteracts the stimulatory effect of HCG on human luteal tissue with high specificity.

Systemic effects of single hindlimb burn injury on skeletal muscle function and cyclic nucleotide levels in the murine model

This study tested the hypothesis that a single hindlimb burn has both local and distant effects. Male CF 1 anaesthetized mice were given a full thickness scald burn of 3 per cent total body surface area (BSA) by immersion of their left hindlimb in water at 95°C for 5 s. Muscle tension was measured through twitch analysis. Levels of cyclic adenosine 3′–5′ monophosphate (cAMP) and cyclic guanosine 3′–5′ monophosphate (cGMP) were analysed by 125I-radioimmunoassay. Measurements were made in gastrocnemei of the ipsilateral burned and contralateral unburned limbs over a 28-day postburn period. Within 1 week the burned limb showed an increase in both tension and a 100-fold increase in levels of cAMP. However, by the end of the second week muscle tension in the burned limb dropped to one-seventh of control values despite persistence of high levels of cAMP. In contrast, the systemic effects were manifested in the unburned contralateral limb which showed tension to undergo a six-fold compensatory increase at the end of the second week with a 75-fold increase in cAMP. By the end of 4 weeks, tension levels of both burned and unburned limbs were attenuated to one-half control values indicating neuromuscular (NM) dysfunction. Nevertheless, cAMP levels remained elevated in both limbs. Levels of cGMP were reduced throughout the 4-week postburn period. Subsequent to the single hindlimb injury both ipsilateral and contralateral gastrocnemei muscles showed elevated levels of total protein content. Thus, in addition to the local effect of burn on the ipsilateral hindlimb, other systemic mediators are involved which influence contralateral limb tension and cyclic nucleotide metabolism. These findings are important to emerging pharmacological therapies undergoing development to combat NM dysfunction resulting from burn injury.

Cyclic nucleotide levels in human breast cancer and in rat mammary tissues during tumor development

The levels of cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic guanosine 3':5'-monophosphate (cGMP) were studied in dimethylbenz(a)anthracene (DMBA)-induced mammary tumors of Sprague-Dawley rats and in human breast cancer. In the rat carcinomas, these levels were significantly lower than in non-malignant tissues when calculated on the basis of DNA content, but higher (cAMP) or equal (cGMP) when calculated on the basis of weight. In human breast cancer the cyclic nucleotide levels were higher than in non-malignant tissues according to both methods of calculation. No correlation was found in human carcinomas between the cyclic nucleotide levels and mitotic index, nuclear grade, tumor size, or lymph node involvement. The rat tumors were subclassified according to state of differentiation, mitotic index, and state of development. Not all the sub-groups had cAMP levels different from normal values. Differences in cAMP levels between the sub-groups could not be correlated with tumor growth rates and/or mitotic index. Thus, cyclic nucleotides may not be useful in prognosis of breast cancer.

Variations in levels of cAMP, DNA and RNA in Streptomyces alboniger under conditions of aerial mycelium formation and repression

The relationship between endogenous levels of cyclic adenosine 3′,5′-monophosphate (cAMP) and the formation of aerial mycelia was investigated in Streptomyces alboniger under conditions of aerial mycelium formation and repression. The relationship between cellular levels of DNA and RNA and aerial mycelium formation was also investigated. In contrast to cellular differentiation in other Streptomyces, neither variations in cAMP, DNA or RNA levels were found to be associated with the development of aerial mycelia in S. alboniger. The regulation of adenylate cyclase in S. alboniger, however, was found to differ from that of Escherichia coli and related organisms in that glucose raised, rather than lowered, endogenous cAMP levels.

Beta-adrenergic responsiveness of human peripheral lymphocytes after mitogenic transformation with phytohemagglutinin

In vitro transformation of human peripheral blood lymphocytes with the mitogen phytohemagglutinin did not alter the total number of beta-adrenergic binding sites for (±)- 125iodocyanopindolol on the surface of intact cells, whereas binding to membrane fragments of transformed cells appeared to be diminished. In isolated membranes, there was also a marked decrease in basal, fluoride- and hormone-stimulated adenylate cyclase activity after phytohemagglutinin treatment. In whole cells, however, a lowering effect of phytohemagglutinin on levels of cyclic adenosine 3′, 5′-monophosphate was not apparent. The discrepancy between data on intact and broken cells indicates that the transformed cells do not acquire additional beta-adrenergic receptors or catalytic adenylate cyclase as their cell surface expands due to blastogenesis. It is therefore concluded that mitogenic transformation of human peripheral lymphocytes does not cause specific changes in the beta-adrenergic/adenylate cyclase system.

Plasma and urine cyclic nucleotide levels in patients with acute and chronic leukemia

Plasma and urine levels of cyclic adenosine 3′,5′-monophosphate (cAMP) and of cyclic guanosine 3′,5′-monophosphate (cGMP) were measured in 35 normal subjects, in 24 patients with nonneoplastic diseases (iron deficiency anemia, peptic ulcer, and cholelithiasis), and in 50 leukemic patients. The leukemic group included patients with acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, and chronic myelogenous leukemia. All patients were recently diagnosed and untreated, except for 5 patients with blastic transformation of chronic myelogenous leukemia who had been previously treated. There were no significant differences in plasma and urine cyclic nucleotide levels between normal subjects and patients with nonneoplastic diseases. In leukemic patients, plasma and urine cAMP levels were similar to those of normal subjects, whereas plasma and urine cGMP levels were markedly elevated. There were no significant differences in cGMP values between the various types of leukemia. After starting treatment, plasma cyclic nucleotide levels were periodically measured in 21 of the patients with acute leukemia; cGMP levels were normalized in all the 16 subjects who attained complete remission, whereas both cAMP and cGMP levels were apparently unaffected in the patients who did not respond to treatment. This suggests that plasma or urine cGMP could be used as an additional parameter to monitor the patient's response to treatment.

Cigarette smoke exposure in vivo increases cyclic GMP in rat lung.

The enzyme guanylate cyclase is stimulated to produce cycle guanosine 3',5'-monophosphate (GMP) when lung tissue is exposed to cigarette smoke in vitro. These experiments tested whether in vivo exposure in rats to cigarette smoke produces a similar response. Adult rats were anaesthetized with pentobarbital and ventilation with mixtures of air and cigarette smoke at 10 cm H2O inspiratory pressure was achieved after a tracheotomy was performed. Lung tissue samples were taken at intervals during 20 min exposure period and analyzed for levels of cyclic adenosine 3', 5'-monophosphate (AMP) and cycle GMP. Blood carboxyhemoglobin (COHb) levels at 5 min and 15 min of exposure showed high, but sublethal levels of COHb. lung tissue cAMP was unchanged with this exposure, but cGMP levels rose dramatically. Rat lungs showed no changes related to ventilation under similar conditions in the absence of smoke. This observed response of cGMP to cigarette smoke may represent an important pulmonary defense mechanism.

Patterns of cyclic nucleotides in normal and leukaemic human leucocytes.

Because recent observations indicate that metabolism of cyclic nucleotides may be altered in neoplastic cells, the intracellular levels of cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine 3',5'-monophosphate (cGMP) were measured in mononuclear leukaemic and normal human leucocytes. The activities of adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases were also determined. Under basal conditions, cAMP levels were always higher in the normal leucocytes, whilst cGMP levels were of the same order of magnitude in both normal and leukaemic cells, causing the cAMP/cGMP ratios to be significantly lower in leukaemic leucocytes. Leukaemic cells significantly increased cyclic nucleotide levels in response to theophylline, but did not respond to serotonin, carbamylcholine or D,L-isoproterenol. Preincubation of these leucocytes with theophylline produced a detectable cAMP response to D,L-isoproterenol but no cGMP response to serotonin or carbamylcholine was found. Adenylate cyclase and guanylate cyclase were significantly lower in leukaemic than in normal cells, which could largely explain the abnormal cyclic nucleotide pattern found in human leukaemic leucocytes. In our experiments, cAMP phosphodiesterase activity was comparable in normal and leukaemic cells, whereas cGMP phosphodiesterase activity was undetectable inall mononuclear-leucocyte preparations with the methods used.

Variations in the levels of cyclic adenosine 3′:5′-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3t [formula omitted]5′-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn 2+ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K m (1 μ m) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn 2+ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.