Dibutyryl Cyclic Guanosine Research Articles

Nitric oxide post-transcriptionally up-regulates LPS-induced IL-8 expression through p38 MAPK activation.

Nitric oxide (NO(.-)) contributes to vascular collapse in septic shock and regulates inflammation. Here, we demonstrate in lipopolysaccharide (LPS)-stimulated human THP-1 cells and monocytes that NO(.-) regulates interleukin (IL)-8 and tumor necrosis factor alpha (TNF-alpha) by distinct mechanisms. Dibutyryl-cyclic guanosine 5'-monophosphate (cGMP) failed to simulate NO(.-)-induced increases in TNF-alpha or IL-8 production. In contrast, dibutyryl-cyclic adenosine monophosphate blocked NO(.-)-induced production of TNF-alpha (P=0.009) but not IL-8. NO(.-) increased IL-8 (5.7-fold at 4 h; P=0.04) and TNF-alpha mRNA levels (2.2-fold at 4 h; P=0.037). However, nuclear run-on assays demonstrated that IL-8 transcription was slightly decreased by NO(.-) (P=0.08), and TNF-alpha was increased (P=0.012). Likewise, NO(.-) had no effect on IL-8 promoter activity (P=0.84) as measured by reporter gene assay. In THP-1 cells and human primary monocytes treated with actinomycin D, NO(.-) had no effect on TNF-alpha mRNA stability (P>0.3 for both cell types) but significantly stabilized IL-8 mRNA (P=0.001 for both cell types). Because of its role in mRNA stabilization, the p38 mitogen-activated protein kinase (MAPK) pathway was examined and found to be activated by NO(.-) in LPS-treated THP-1 cells and human monocytes. Further, SB202190, a p38 MAPK inhibitor, was shown to block NO(.-)-induced stabilization of IL-8 mRNA (P<0.02 for both cell types). Thus, NO(.-) regulates IL-8 but not TNF-alpha post-transcriptionally. IL-8 mRNA stabilization by NO(.-) is independent of cGMP and at least partially dependent on p38 MAPK activation.

PGE 2 and dibutyryl cyclic adenosine monophosphate prolong eosinophil survival in vitro

Background: Apoptosis represents a mechanism by which the accumulation and inflammatory potential of eosinophils in asthma might be limited. Mediators derived from the airway epithelium may influence the rate of eosinophil apoptosis. Objective: We have investigated the effects on eosinophil apoptosis of 3 mediators that are likely to be produced by the airway epithelium, namely PGE 2 , TNF-α, and nitric oxide. Methods: Peripheral blood eosinophils from healthy adult volunteers were purified by density gradient centrifugation and negative immunomagnetic selection. Eosinophils were cultured for 16 or 40 hours with PGE 2 (10 nmol/L), dibutyryl cyclic adenosine monophosphate (AMP; 100 μmol/L), TNF-α (500 U/mL), the nitric oxide donors, S-nitroso-N-acetylpenicillamine (100 μmol/L), and 2,2 ́-(hydroxynitrosohydrazono)bis-ethanamine (1 mmol/L), or dibutyryl cyclic guanosine monophosphate (100 μmol/L). Control cultures consisted of untreated, IL5–treated (100 U/mL), and anti–Fas-treated (400 ng/mL) cells. Eosinophil apoptosis was assessed by flow cytometric analysis of annexin V-FITC binding to externalized phosphatidylserine, by electrophoresis of phosphorus 32 end-labeled DNA fragments, and by flow cytometric assessment of hypodiploid DNA with propidium iodide. Results: PGE 2 and cyclic AMP inhibited spontaneous eosinophil apoptosis at both 16 and 40 hours as did the PGEP 2 receptor agonist, 11-deoxy PGE 1 , at 40 hours, but these effects were not inhibited by a protein kinase A antagonist. TNF-α delayed apoptosis in eosinophil cultures at 16 hours, whereas S-nitroso- N-acetylpenicillamine, 2,2 ́-(hydroxynitrosohydrazono)bis-ethanamine, and cyclic guanosine monophosphate had little effect. Anti-Fas had little effect on spontaneous eosinophil apoptosis but significantly reduced the inhibitory effects of PGE 2 , cyclic AMP, and TNF-α. Assessments of apoptosis by DNA fragmentation gave similar but quantitatively less sensitive results. Conclusion: Inhibition of spontaneous eosinophil apoptosis by PGE 2 appears to be mediated by EP 2 receptors but is not protein kinase A dependent. By enhancing eosinophil survival, PGE 2 may increase the proinflammatory potential of these cells in chronic asthma. (J Allergy Clin Immunol 1999;104:153-62.)

Role of cyclic nucleotides in pigment translocation within the freshwater shrimp, Macrobrachium potiuna , erythrophore

The participation of cyclic nucleotide-dependent intracellular signalling pathways in the pigment translocation induced by pigment-dispersing hormone (α -PDH) or pigment-concentrating hormone (PCH) was investigated in the erythrophores of the freshwater shrimp, Macrobrachium potiuna. Cholera toxin, forskolin and dibutyryl cyclic adenosine 3′5′ monophosphate (dbcAMP) were able to induce pigment dispersion with effective agonist concentrations for half maximal response (EC50 s) of 2.8 · 10−11 mol · l−1, 7.0 · 10−7 mol · l−1 and 3.3 · 10−7 mol · l−1, respectively. KT5720 (10−7 mol · l−1 and 10−6 mol · l−1) significantly shifted the dose response curve to α -PDH to the right. Dibutyryl cyclic guanosine 3′5′ monophosphate (dbcGMP) was ineffective in inducing either pigment aggregation or dispersion. 2′5′ dideoxyadenosine (DDA) and SQ22,536 essentially elicit a pigment-aggregating response in a dose-dependent manner. These effects were not due to the activation of purinergic receptors, since concentrations up to 10−4 mol · l−1 of adenosine and adenosine triphosphate (ATP), and up to 10−3 mol · l−1 of uracil triphosphate (UTP) did not elicit pigment aggregation. In order to verify if PCH decreased cyclic adenosine 3′5′ monophosphate (cAMP) levels, cumulative dose-response curves to PCH in the absence and presence of pertussis toxin and 8-MOM-IBMX were determined. However, neither drug significantly affected PCH activity. The levels of cAMP in the integument cells of M. potiuna were significantly increased (P < 0.05) by α -PDH (10−7 mol · l−1) and forskolin (10−6 mol · l−1), but were not affected by PCH (10−7 or 10−10 mol · l−1). In conclusion, α -PDH seems to elicit pigment dispersion through the activation of a Gs-protein coupled receptor resulting in cAMP increase and cAMP-dependent protein kinase (PKA) activation. Furthermore, although a decrease in cAMP was assumed to be responsible in turn for the action of PCH, such a decrease could not be directly demonstrated.

Nitric oxide is not involved in the initiation of insulin secretion from rat islets of Langerhans.

The involvement of nitric oxide as an intracellular messenger in the control of insulin secretion from pancreatic Beta cells was studied in rat islets of Langerhans by measuring: (i) nitric oxide generation in response to physiological insulin secretagogues; (ii) the effects of inhibitors of nitric oxide synthesis on insulin secretory responses to physiological secretagogues, and on insulin synthesis; (iii) changes in islet cyclic guanosine monophosphate in response to secretagogues; (iv) the effects of exogenous cyclic guanosine monophosphate and dibutyryl cyclic guanosine monophosphate on insulin secretion from electrically permeabilised islets and from intact, respectively. These studies produced no evidence that nitric oxide generation is required for the initiation of insulin secretion by common secretagogues. However, the results of our experiments suggest that the generation of nitric oxide may be involved in long-term, glucose-dependent increases in cyclic guanosine monophosphate content of islet cells, although the physiological relevance of these changes requires further investigation.

Cyclic AMP potentiates bFGF-induced neurite outgrowth in PC12 cells.

We report here that basic fibroblast growth factor (bFGF)-elicited neurite outgrowth in PC12 cells is potentiated by dibutyryl cyclic adenosine monophosphate (dbcAMP) or forskolin. This property was also described for nerve growth factor (NGF), suggesting that both NGF and bFGF may share common intracellular events leading to neurite outgrowth and synergism with dbcAMP and forskolin. The synergistic effect of dbcAMP and forskolin is specific, since treatment of PC12 cells with bFGF and dibutyryl cyclic guanosine monophosphate (dbcGMP) or phorbol ester did not change the neurite outgrowth response of cells treated with bFGF alone. Furthermore, neurite outgrowth depends on cellular adhesion. Increasing adhesion by plate treatment with poly-d-lysine increases the neurite outgrowth elicited by bFGF alone or bFGF plus dbcAMP. On the other hand, decreasing cellular adhesiveness by plating PC12 cells in semi-solid agarose renders the cells unable to develop neuritic processes. In addition, 3H-methylthymidine incorporation studies showed that bFGF-treated PC12 cells cease growth only when they become fully differentiated after 3-5 days of treatment. In contrast, dbcAMP, which is a poor differentiation factor, is able to block cellular growth after 24 hour treatment. These results suggest that when PC12 cells become differentiated, they stop growing. However, growth inhibition does not necessarily lead to differentiation.

Effects of dibutyryl cyclic guanosine monophosphate on human spermatozoal motility and penetration of zona-free hamster oocytes

The possible effects of N2,2'-O-dibutyryl 3'-5'-cyclic guanosine monophosphate (dbcGMP), imidazole (IM) or their combination on human spermatozoal motility and penetration of zona-free hamster oocytes were investigated. Motility of human spermatozoa was assessed at 0, 0.5, 2, 3, 5 and 8 h over an 8-h incubation period with various concentrations of dbcGMP, IM or dbcGMP plus IM. Both percentage motile spermatozoa and progression of human spermatozoa decreased with increasing concentrations of dbcGMP (12-96 mM) and IM (10-80 mM). This inhibitory effect on motility was more pronounced when dbcGMP was added with IM. For assessment of human spermatozoal penetration into zona-free hamster oocytes, spermatozoa were treated for 30 min with either dbcGMP, IM or both after 5 h of capacitation pre-incubation at 37 degrees C. Alternatively, the compounds were only added during the capacitation preincubation period. At concentrations of dbcGMP less than or equal to 24 mM, neither of the treatments had a significant effect on the penetration rate of zona-free hamster oocytes in comparison with the controls. However, a higher concentration of dbcGMP (48 mM) inhibited the penetration rate. A similar trend was observed when dbcGMP plus IM were added. IM alone had no significant effect on the penetration rate at the concentrations tested (10-40 mM). Our data indicated an inhibitory effect of dbcGMP on both human spermatozoal motility and penetration into zona-free hamster oocytes.

Control of Bovine Uterine Prostaglandin F2α Release in Vitro1

Prostaglandin F2 alpha (PGF2 alpha) release from the uterus causes luteolysis in ruminants, and oxytocin is thought to be a regulator of this release. In the present study, we have examined the mechanisms involved in oxytocin stimulation of PGF2 alpha secretion by bovine endometrium in vitro. Endometrial tissue explants, obtained from heifers at Day 19 or 20 (n = 3) and Day 0 (estrus, n = 5) of the estrous cycle, were incubated for 2 h and 6 h, and PGF2 alpha concentration in the medium was determined by radioimmunoassay (RIA). Basal PGF2 alpha release increased for up to 6 h and was significantly stimulated after 2 h of incubation with 100 microU and 1000 microU of oxytocin at Day 0 but not at Day 19 or 20. Secretion of PGF2 alpha was not affected by cholera toxin (10 ng/ml) or the cyclic nucleotide analogs dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate at a concentration of 1 mM. A protein kinase A inhibitor (500 microM) had no effect on the oxytocin-induced release of PGF2 alpha. Both the phorbol ester, 12-myristate-13-acetate (100 mM), and the non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol (500 microM), significantly stimulated PGF2 alpha secretion to the same extent as oxytocin. Neither basal nor stimulated PGF2 alpha release was affected by the calcium ionophore A23187 (0.1-5.0 microM). However, PGF2 alpha secretion was sensitive to cycloheximide (1 microgram/ml) suggesting that protein synthesis may be involved. In conclusion, these data suggest that the stimulation of PGF2 alpha by oxytocin is via the protein kinase C effector pathway.

Effects of N6, 2‘‐0‐dibutyryladenosine 3’,5‘‐cyclic monophosphate, adenosine, and of oxotremorine and 3‐isobutyl‐1‐methylxanthine on the electrically evoked [3H] acetylcholine secretion in the guinea‐pig ileum myenteric plexus

The guinea-pig ileum longitudinal muscle-myenteric plexus preparation, pre-incubated with [3H]choline, was mounted in an organ bath and superfused with Tyrode's solution. [3H]Acetylcholine secretion was evoked by 150 electrical shocks at 0.5 Hz. N6,2'-O-Dibutyryladenosine 3',5'-cyclic monophosphate (dibutyryl cyclic AMP) enhanced the [3H]acetylcholine secretion in the presence of eserine and the adenosine receptor antagonist 8-phenyltheophylline (10 mumol l-1). Conversely, in the absence of 8-phenyltheophylline the [3H]acetylcholine secretion was reduced by dibutyryl cyclic AMP. In the absence and presence of 8-phenyltheophylline (apparent KD = 12 mumol l-1), adenosine reduced the [3H]acetylcholine secretion to 33% of control (IC50 = 8 mumol l-1) and to 48% of control (IC50 = 14 mumol l-1) respectively. Neither butyrate, dibutyryl cyclic GMP nor guanosine altered the [3H]acetylcholine secretion. Interaction experiments with 3-isobutyl-1-methylxanthine and oxotremorine were done in the absence of eserine, i.e. when oxotremorine is effective. Oxotremorine depressed the fractional secretion of [3H]acetylcholine with a 'maximal inhibition' of 13% of control (IC50 = 10 nmol l-1). In the presence of 3-isobutyl-1-methylxanthine (5 mmol l-1) oxotremorine depressed the secretion to 2% of control with an apparent IC50 value of 0.9 mumol l-1. 3-Isobutyl-I-methylxanthine (0.01-4 mmol l-1) enhanced the fractional secretion of [3H]acetylcholine with a 'maximal enhancement' value of 232% of control (EC50 = 0.19 mmol l-1). The presence of oxotremorine (30 nmol l-1) counteracted, and higher concentrations reversed, the enhancement caused by 3-isobutyl-1-methylxanthine.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of chemotaxis Ng-monomethyl-L-arginine: a role for cyclic GMP

The metabolism of L-arginine to nitric oxide (NO) has been shown to be important for the effector functions of many cell types, including polymorphonuclear (PMN) leukocytes. Its effect appears to be mediated at least in part by NO stimulation of soluble guanylate cyclase. We evaluated the role of this pathway in two PMN effector functions: cell movement and microbial killing, using the competitive inhibitor of L- arginine conversion to NO, NG-monomethyl-L-arginine (NMA). We also evaluated the effect of additional L-arginine and dibutyryl cyclic guanosine monophosphate (cGMP) on any NMA-associated changes. Human peripheral blood neutrophils were used and the cells were incubated with and without NMA. Chemotaxis was evaluated using a 48-well micro- Boyden chamber. Microbial killing was evaluated using S aureus strains D2C and 502A. These studies demonstrated that chemotaxis to formyl- methionyl-leucyl-phenylalanine was markedly inhibited in NMA-treated cells. This inhibition could be overcome if L-arginine or dibutyryl cGMP were added with the NMA. In contrast, microbial killing of S aureus was unaffected by NMA. These observations support the hypothesis that the L-arginine metabolism to NO and its effect on the cGMP level may be important for the dynamic changes required for neutrophil chemotaxis.

Inhibition of Chemotaxis With NG-Monomethyl-L-Arginine: A Role for Cyclic GMP

The metabolism of L-arginine to nitric oxide (NO) has been shown to be important for the effector functions of many cell types, including polymorphonuclear (PMN) leukocytes. Its effect appears to be mediated at least in part by NO stimulation of soluble guanylate cyclase. We evaluated the role of this pathway in two PMN effector functions: cell movement and microbial killing, using the competitive inhibitor of L-arginine conversion to NO, NG-monomethyl-L-arginine (NMA). We also evaluated the effect of additional L-arginine and dibutyryl cyclic guanosine monophosphate (cGMP) on any NMA-associated changes. Human peripheral blood neutrophils were used and the cells were incubated with and without NMA. Chemotaxis was evaluated using a 48-well micro-Boyden chamber. Microbial killing was evaluated using S aureus strains D2C and 502A. These studies demonstrated that chemotaxis to formyl-methionyl-leucyl-phenylalanine was markedly inhibited in NMA-treated cells. This inhibition could be overcome if L-arginine or dibutyryl cGMP were added with the NMA. In contrast, microbial killing of S aureus was unaffected by NMA. These observations support the hypothesis that the L-arginine metabolism to NO and its effect on the cGMP level may be important for the dynamic changes required for neutrophil chemotaxis.

Cholecystokinin-octapeptide constricts guinea-pig and human airways.

1. Cholecystokinin-octapeptide (CCK-OP, 10(-10)-3 x 10(-6) M) produced a concentration-dependent contractile response in guinea-pig trachea which was enhanced by both the mechanical removal of the epithelium and by indomethacin (10(-5) M), with an EC50 of 6.18 +/- 0.10 x 10(-8) M. 2. Sub-threshold concentrations of CCK-OP, which did not alter the resting tone of the smooth muscle, did not alter responses produced to electrical field stimulation (EFS) or to vagal nerve stimulation in an intact tracheal tube preparation. Atropine (2 x 10(-6) M) did not alter the concentration-response curve to CCK-OP, indicating that CCK-OP contraction is not mediated by cholinergic mechanisms. 3. The inhibition of neutral endopeptidase (endopeptidase-24.11) by phosphoramidon (10(-5) M) gave a leftward shift in the CCK-OP concentration-response curve in tissues with intact epithelium obtained from normal animals, but had no effect in tissues denuded of epithelium or in tissues obtained from animals which had been actively sensitized and challenged with ovalbumin (OA). 4. CCK-OP-induced contractile responses were antagonized by the CCK-receptor antagonists dibutyryl cyclic guanosine monophosphate (pA2 = 4.3) and L-364,718 (pA2 = 9.6). 5. CCK-OP induced bronchoconstriction in large, but not small, human airways and was antagonized by the CCK-receptor antagonist L-364,718. CCK-OP had no effect on cholinergic neural responses elicited by EFS in human airways.

Cyclic nucleotides regulate collagen production by human intestinal smooth muscle cells

The effect of cyclic nucleotides on collagen production by human intestinal smooth muscle cells was examined in vitro. Cholera toxin and isobutyl-methylxanthine, agents that elevate cyclic adenosine monophosphate, caused selective inhibition of collagen production when cells were exposed to these agents for 24–72 h. Exposure for 6 h inhibited noncollagen protein synthesis without effects on collagen production. Forskolin similarly inhibited collagen production, decreasing relative collagen synthesis 40% at 10 μM and 60% at 100 μM. After 48 h of exposure to cholera toxin and isobutylmethylxanthine, levels of cyclic adenosine monophosphate had increased in a concentration-dependent manner. The effect of cyclic nucleotide analogues was also examined. Dibutyryl cyclic adenosine monophosphate inhibited collagen production, whereas dibutyryl cyclic guanosine monophosphate increased collagen production by 65%. This effect was maximal at a concentration of 10 μM. These observations suggest that cyclic adenosine monophosphate has a significant effect on collagen production by human intestinal smooth muscle cells in vitro and may play a role in the modulation of collagen production by these cells in vivo.

Fertilization through spermatozoal microinjection: significance of acrosome reaction.

Acrosome-reacted spermatozoa were microinjected into the perivitelline space of mouse oocytes. After 2 h incubation in culture medium containing lactate and albumin, spermatozoa were transferred into culture medium containing 12 mM of dibutyryl cyclic guanosine 3',5'-monophosphate (dbcGMP) and 10 mM imidazole for 20 min. One motile spermatozoon was injected into the perivitelline space of each oocyte. Fertilization was recognized by the presence of a second polar body and two pronuclei. The overall fertilization rate was 19.6% in the case of dbcGMP-treated spermatozoa as compared to 5.3% for non-treated spermatozoa. Thus, acrosome-reacted motile spermatozoa improve the fertilization rate of sperm microinjection. Sperm microinjection may be a method to foster fertility in cases of oligo-/asthenozoospermia in human in-vitro fertilization.

Pharmacological inhibition of interleukin-1 activity on T cells by hydrocortisone, cyclosporine, prostaglandins, and cyclic nucleotides

The effects of a panel of hormones and pharmacological agents on the activation of T cells by a combination of interleukin-1 and phytohemagglutinin (IL-1/PHA) was studied. Pharmacological effects on various stages of IL-1/PHA-induced interleukin-2 (IL-2) production by the cloned murine thymoma cell line LBRM-33-1A5.7 were dissected using a multi-step assay procedure. A 4-h lag phase in the kinetics of IL-2 production allowed the operational definition of an early, IL-1-dependent programming stage, followed by an IL-2-production stage of the assay. A cell-washing procedure between these stages was introduced in order to distinguish IL-1 receptor antagonists from functional IL-1/PHA antagonists. Hydrocortisone and cyclosporine were potent inhibitors (active in the nM range) of both stages of IL-2 production, suggesting that neither is an IL-1 receptor antagonist. The cyclic adenosine monophosphate (cAMP)-elevating agents prostaglandin E2, dibutyryl cAMP, and theophylline inhibited IL-2 production during the early, IL-1-dependent programming stage. By contrast, prostaglandin F2α and dibutyryl cyclic guanosine monophosphate did not appreciably inhibit IL-1/PHA activity. These results are discussed in relationship to the effects of these test agents in thymocyte IL-1 assays or mitogenesis assays and the implications toward understanding the mechanisms underlying IL-1/PHA activation of T cells.

Inhibition of Ornithine Decarboxylase Activity by Follicle Stimulating Hormone in Primary Culture of Rat Sertoli Cells

The effect of follicle stimulating hormone on the activity of ornithine decarboxylase (ODC) was determined in primary culture of rat Sertoli cells. Three different FSH preparations (NIH oFSH-S-15, S-16, and eFSH) inhibited ODC activity in rat Sertoli cells under different media conditions. The inhibition was both time- and dose-dependent. The mechanism of the FSH inhibitory effect was studied using dibutyryl cyclic adenosine monophosphate (dbcAMP), 1-methyl-3-isobutylxanthine (MIX), forskolin, and isoproterenol. All of these agents, known to elevate cellular cAMP levels, inhibited ODC activity in cultured rat Sertoli cells. The combined effect of each of these substances plus FSH was either greater than, or equal to, that of FSH alone, and was not additive. Dibutyryl cyclic guanosine monophosphate had no effect on the ODC activity. These findings suggest that FSH inhibition of ODC activity in the rat Sertoli cell may be mediated by cAMP.

Ion pump ATPase inhibitors block the fertilization of zona-free mouse oocytes by acrosome-reacted spermatozoa

The acrosome reaction was induced in nearly 100% of mouse spermatozoa with dibutyryl cyclic guanosine monophosphate (dbcGMP) before ouabain treatment. Acrosome-reacted spermatozoa could not penetrate the zona pellucida, but readily fertilized zona-free eggs. Exposure to ouabain at concentrations as low as 10(-7) M had a noticeable inhibitory effect upon fertilization. Similar results were obtained with a second ATPase inhibitor, digoxin. These results show that ion-pump inhibitors block the union of gametes which are otherwise fully competent to fertilize. These findings suggest that a membrane potential maintained by ion pumps is a necessary prerequisite for gamete fusion.

Pharmacologic characterization of excitatory and inhibitory cholecystokinin receptors of the cat gallbladder and sphincter of Oddi

The pharmacologic properties and specificity of cholecystokinin (CCK) receptors of the biliary tract were investigated by testing various sulfated and desulfated CCK fractions and by using specific CCK-gastrin antagonists. Sulfated CCK-7 (5–80 pmol/kg) caused gallbladder contraction and sphincter of Oddi relaxation. Denervation with tetrodotoxin decreased the gallbladder response by 50% and changed the sphincter of Oddi response from relaxation to contraction. Desulfated CCK-7 (80–400 pmol/kg) caused a weak gallbladder contraction that was unaffected by tetrodotoxin. The gallbladder did not respond to CCK-3 (10–80 nmol/kg) or to CCK-2 (10–160 nmol/kg) in doses that completely relaxed the sphincter of Oddi. These doses, however, were 5–2000 times higher than the maximal dose of sulfated CCK-7. After denervation with tetrodotoxin, desulfated CCK-7 (10–400 pmol/kg) induced a weak sphincter of Oddi contraction even with doses five times greater than the maximal dose of sulfated CCK-7. The denervated sphincter of Oddi did not respond to CCK-3 (10–80 nmol/kg) or CCK-2 (10–160 nmol/kg). Furthermore, a continuous proglumide infusion (5–20 mg/kg · min) and bolus doses of dibutyryl cyclic guanosine monophosphate (250–1000 μg/kg) blocked the effect of sulfated CCK-8 on the gallbladder and sphincter of Oddi. Higher doses of these antagonists were needed, however, to block the CCK effect on the sphincter of Oddi than on the gallbladder. In contrast, high doses of desulfated CCK-7 (100 pmol/kg) or CCK-3 (200 nmol/kg) did not antagonize the effect of sulfated CCK-8 (10–80 pmol/kg) on the gallbladder. These findings suggest the existence of three sets of specific CCK receptors with molecular configuration requirements determined by the type of cell where these receptors are located: on the postganglionic cholinergic neurons, on the smooth muscle cells of the gallbladder, and sphincter of Oddi, or on the postganglionic noncholinergic, nonadrenergic inhibitory neurons.

An experimental trial of cyclic nucleotides on multicellular spheroids derived from human brain tumours.

The effects of cyclic nucleotides, dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate (db-cAMP and db-cGMP), on the growth rate of multicellular tumour spheroids were evaluated by comparing the growth delay and colony forming efficiency in vitro. Multicellular tumour spheroids were derived directly from human brain tumours. To compare the chemotherapeutic effect of cyclic nucleotides, CCNU was used as a known effective cytotoxic drug on malignant gliomas. Significant growth delay was obtained by db-cAMP (p less than 0.001) while CCNU was tumouricidal rather then producing a delay in growth of the tumour spheroids. Db-cGMP found not to be effective in decreasing the growth rate of the tumour spheroids in vitro (p greater than 0.2). The role of cyclic nucleotides in brain tumours is discussed on a review basis.

Effects of dibutyryl cyclic adenosine monophosphate and dibutyryl cyclic guanosine monophosphate on neuron activity of suprachiasmatic nucleus in rat hypothalamic slice preparation

In order to elucidate the role of cyclic nucleotides in the suprachiasmatic nucleus, we examined the effect of dibutyryl cyclic AMP (c-AMP) and dibutyryl cyclic GMP (c-GMP) on single neuronal activity of the suprachiasmatic nucleus of the rat. A hypothalamic slice about 300 μm thick, including the suprachiasmatic nucleus, was cut coronally using a vibratome. By means of bath- as well as iontophoretic application, c-AMP and c-GMP caused mainly an inhibition and facilitation in suprachiasmatic neuronal activity, respectively. The present results suggest that c-AMP and c-GMP play an important role in regulation of suprachiasmatic neuronal activity as a second messenger and that these two nucleotides may function as a reciprocal messenger.

The effect of cyclic nucleotides on secretin secretion in canine duodenal mucosa in vitro

We examined the effects of cyclic nucleotides and calcium on secretin release from canine duodenal mucosal explants incubated in organ culture media. Time course studies revealed that at pH 7.4, 5 and 10 mM dibutyryl cyclic adenosine monophosphate (DBcAMP) increased secretin release progressively, reaching a peak at 2 hours. Two mM of DBcAMP at pH 7.4 did not increase secretin release but at pH 4.5, all 3 doses potentiated secretin release. DBcAMP-stimulated secretin release was not dependent on the influx of extracellular calcium. Graded doses of 3-isobutyl-1-methylxanthine (IBMX) did not stimulate secretin secretion but 1 mM IBMX with 2 mM DBcAMP increased secretin secretion significantly. Dibutyryl cyclic guanosine monophosphate, cholera toxin and 5′-guanylyl-imidodiphosphate (GPP(NH)p) did not stimulate basal secretin release. The release of secretin from our explants incubated at pH 7.4 was not due to specific leakage because all of our viability studies revealed that our explants were functionally intact at the end of 2 hours. Our observations suggest that cyclic nucleotides may participate in the intracellular regulation of secretin secretion.