Db Cyclic AMP Research Articles

A re-assessment of a combinatorial treatment involving Schwann cell transplants and elevation of cyclic AMP on recovery of motor function following thoracic spinal cord injury in rats

This study was undertaken as part of the NIH “Facilities of Research-Spinal Cord Injury” project to support independent replication of published studies. Here, we repeated a study reporting that a combinatorial treatment with transplants of Schwann cells, systemic delivery of Rolipram to enhance cyclic AMP levels, and intra-spinal injections of dibutyryl cyclic AMP enhanced locomotor recovery in rats after contusion injuries at the thoracic level. We compared the following experimental groups: 1) rats that received Schwann cell transplants, systemic Rolipram, and injections of db-cyclic AMP (the combined treatment group that showed the greatest improvement in function); 2) rats that received Schwann cell transplants only and implantation of empty pumps as control; 3) rats that received Rolipram only and implantation of empty pumps as control, and 4) control rats that received no treatment other than the injection of DMEM into the spinal cord and implantation of empty pumps. The principal findings reported in Pearse et al. were not replicated in that the combined treatment group did not exhibit greater recovery on any of the measures, although the group that received Schwann cells only did exhibit enhanced recovery on several of the outcome measures. The failure of the combined treatment may be due in part to less successful engraftment of Schwann cells in our study vs. Pearse et al. Issues relating to failures to replicate, especially when effect size is small, are discussed.

Brain Control of Mating Behavior in the Male Cricket Gryllus bimaculatus DeGeer: Excitatory Control of Copulatory Actions

To understand the functional role of the insect brain in mating behavior, copulatory actions in response to a model stimulus were compared between intact and decerebrated male crickets. Decerebrated males were then tested to examine whether their copulatory actions were modifed by biogenic amines or electrical stimulation. The main difference in copulatory actions between intact and decerebrated males was in the body thrusts consisting of the protraction and retraction of the abdomen for hooking. These movements became slower after the removal of the brain, as measured by the average interval between responses, called the cycle length. The cycle length increased to twice the length in intact males within about 20 min. Intraperitoneal injection of octopamine in decerebrated males shortened the cycle length dose-dependently (10−6–10−2M) and restored it nearly to the level in intact males at 10−4–10−2M. Octopamine-mimicking agents forskolin, IBMX and synephrine, and cyclic AMP analogue DB-cyclic AMP had effects similar to that of octopamine, while serotonin, dopamine, noradrenaline and adrenaline did not. Electrical stimulation of the neck connectives mimicked the effect of octopamine, which was blocked by octopamine antagonist phentolamine. Perfusion of the hemocoel with Ringer's solution to eliminate the octopamine previously injected abolished the effect of extrinsic octopamine, whereas it did not abolish the effect of electrical stimulation. These results suggest that the brain in the male cricket is involved in facilitating the activity of the pattern generator for mating behavior via intraganglionic octopamine.

Glomerular effects of cholera toxin in isolated perfused rat kidney: a potential role for platelet activating factor.

Cholera toxin (MW 84 kDa) is now considered a pharmacological tool to study the adenylyl cyclase system and a stimulus to generate platelet activating factor in the intestinal tract. We used this toxin to evaluate the renal haemodynamics, glomerular filtration function, tubular sodium transport and toxicity in isolated perfused rat kidney. Kidneys from adult male Wistar rats were isolated for perfusion. The perfusion fluid was modified Krebs-Henseleit solution and the samples were analyzed for sodium, potassium, inulin and osmolality. Clearance techniques were used to calculate physiological parameters. Cholera toxin (1.0 microg/ml) caused a significant time-dependent reduction of glomerular filtration rate and urinary flow. This toxin also caused a small, but consistent reduction in fractional proximal sodium reabsortion (toxin = 67.43+/-2.42% versus control = 79.26+/-5.80%; P<0.025). WEB 2086, a platelet activating factor receptor antagonist at 100 microg/ml completely blocked the effects induced by cholera toxin on glomerular filtration rate, fractional proximal sodium reabsortion and urinary flow. In contrast to cholera toxin, dibutyryl-cyclic AMP (10(-5) M) significantly increased glomerular filtration rate (Db-cyclic AMP = 0.651+/-0.035 versus control = 0.514+/-0.043 ml x g(-1) x min(-1); P<0.025) in isolated perfused kidneys. Db-cyclic AMP caused a similar, but more severe reduction in fractional proximal sodium reabsortion (Db-cyclic AMP = 54.21+/-2.35% versus control = 70.10+/-3.24%; P<0.025). In addition Db-cyclic AMP increased significantly the urinary flow (Db-Cyclic AMP = 0.290+/-0.018 versus control = 0.179+/-0.026 ml x g(-1) x min.(-1); P<0.025). WEB 2086+ Db-cyclic AMP also caused a significant increase in the urinary flow with maximal effect at 90 min. (WEB+Db-cyclic AMP = 0.26+/-0.01 versus control = 0.15+/-0.01 ml x g(-1) x min.(-1); n = 8, P<0.025). Cholera toxin caused a decrease of urinary flow (toxin = 0.034+/-0.004 versus control = 0.145+/-0.02 ml x g(-1) x min.(-1); P<0.025), this effect was also completely abolished by WEB 2086 when it was injected previously to toxin. When only WEB 2086 was injected, the functional parameters remained stable throughout the perfusion time. Cholera toxin had no effect on renal vascular resistance, renal perfusate flow or tissue potassium, suggesting renal integrity in kidneys treated with this toxin. The results suggest that cholera toxin effects in the perfused rat kidney are primarily mediated by platelet activating factor.

Second messengers and the control of progesterone production from first trimester trophoblast.

Basal progesterone production from first trimester placental cells in culture was high during the first 24 h of culture and fell to less than 30% of the initial level after 96 h in vitro. 22(R)-Hydroxycholesterol had a similar effect on progesterone production at all incubation times, indicating that the decline in basal steroidogenesis was not due to a loss of mitochondrial or post-mitochondrial enzymes. Continuous stimulation with dibutyryl (db) cyclic AMP maintained progesterone synthesis at a relatively constant high level despite the fall in basal progesterone production, and the optimum concentration of db cyclic AMP was 1.0 mM. The calcium ionophore A23187 had no effect on progesterone incubation during short-term cultures (<4 h), and inhibited steroidogenesis after 24 h. Repeated addition of A23187 during 96 h of culture also inhibited progesterone production. These findings indicate that progesterone production in human trophoblast is supported by a local factor which maintains a high level of steroid production through a cyclic AMP-dependent mechanism. The inhibitory effects of calcium ionophore in trophoblast differ from the stimulatory effects of this compound in other steroidogenic cells, but the reasons for the difference are not known at present.

Baclofen reduces GABAA receptor responses in acutely dissociated neurons of bullfrog dorsal root ganglia

The effect of baclofen on the function of the γ-aminobutyric acidA (GABAA) receptor was examined in acutely dissociated neurons of bullfrog dorsal root ganglia (DRG) by using the whole-cell coltage-clamp method. Baclofen (0.1–100 μM) depressed the inward currents produced by GABA (100 μM) and muscimol (100 μM). Baclofen shifted the concentration-response curve for GABA (1 μM-1 mM) downward. Baclofen decreased the maximum response (Vmax) to GABA without changing the apparent dissociation constant (Kd), suggesting a noncompetitive antagonism. The effect of baclofen on the GABA current was blocked by antagonists for the GABAB receptor; the rank order of potency was P-[3-Aminopropyl]-P-diethoxymethylphosphinic acid (CGP 55845A) ≫ 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-benzylphosphinic acid (CGP 35348) > saclofen ≫ phaclofen. Baclofen produced an irreversible depression of the GABA current in neurons dialyzed with an internal solution containing guanosine (5′-O-(3-thiotriphosphate) (GTPγS, 100 μM). Intracellular guanosine 5′-O-(2-thiodiphosphate) (GDPβS, 100 μM) blocked the inhibitory effect of baclofen on the GABA current. Forskolin (10 μM) and dibutyryl N6, 2′-O-dibutyryladenosine 3′:5′-cyclic monophophate (db-cyclic AMP) (200 μM) depressed the GABA current. N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9, 40 μM) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 50 μM), protein kinase A (PKA) inhibitors, reduced the depressant effect of baclofen on the GABA current. The baclofen-induced depression of the GABA current was blocked by PKI(5–24), a specific PKA inhibitor, but not by PKC(19–36), a specific protein kinase C(PKC) inhibitor. We suggest that GABAB receptors regulate the GABAA receptor function through a G-protein linked to the adenylyl cyclase-PKA pathway in bullfrog DRG neurons. Synapse 26:165–174, 1997. © 1997 Wiley-Liss, Inc.

Baclofen reduces GABAA receptor responses in acutely dissociated neurons of bullfrog dorsal root ganglia.

The effect of baclofen on the function of the gamma-aminobutyric acidA (GABAA) receptor was examined in acutely dissociated neurons of bullfrog dorsal root ganaglia (DRG) by using the whole-cell voltage-clamp method. Baclofen (0.1-100 microM) depressed the inward currents produced by GABA (100 microM) and muscimol (100 microM). Baclofen shifted the concentration-response curve for GABA (1 microM-1 mM) downward. Baclofen decreased the maximum response (Vmax) to GABA without changing the apparent dissociation constant (Kd), suggesting a noncompetitive antagonism. The effect of baclofen on the GABA current was blocked by antagonists for the GABAB receptor; the rank order of potency was P-[3-Aminopropyl]-P-diethoxymethylphosphinic acid (CGP 55845A) > > 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P- benzyl-phosphinic acid (CGP 35348) > saclofen > > phaclofen. Baclofen produced an irreversible depression of the GABA current in neurons dialyzed with an internal solution containing guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S, 100 microM). Intracellular guanosine 5'-O-(2-thiodiphosphate) (GDP beta S, 100 microM) blocked the inhibitory effect of baclofen on the GABA current. Forskolin (10 microM) and dibutyryl N6, 2'-O-dibutyryladenosine 3':5'-cyclic monophophate (db-cyclic AMP) (200 microM) depressed the GABA current. N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9, 40 microM) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004, 50 microM), protein kinase A (PKA) inhibitors, reduced the depressant effect of baclofen on the GABA current. The baclofen-induced depression of the GABA current was blocked by PKI(5-24), a specific PKA inhibitor, but not by PKC(19-36), a specific protein kinase C (PKC) inhibitor. We suggest that GABAB receptors regulate the GABAA receptor function through a G-protein linked to the adenylyl cyclase-PKA pathway in bullfrog DRG neurons.

Cyclic AMP-mediated enhancement of high-affinity choline transport and acetylcholine synthesis in brain.

Intracerebroventricular administration of N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cyclic AMP) to mice increased high-affinity choline transport (HAChT) into synaptosomal preparations from the hippocampus, striatum, and frontal cortex in a time- dose-, and brain region-dependent manner. Similar observations were made when the cyclic AMP analogue 8-bromo-cyclic AMP, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine were administered. Inhibition of phosphatase 1 and 2A, with okadaic acid, increased basal choline transport and enhanced the response to db-cyclic AMP. The early increase of HAChT activity induced by db-cyclic AMP was blocked by H-7 and H-89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent Vmax without a change of the Km for choline. Hemicholinium-3 (HC-3) binding was not altered when studied 1 h after db-cyclic AMP administration. In contrast, HC-3 binding and HAChT activity were both elevated when estimated 3 h after the treatment, and pretreatment with cycloheximide partially prevented the db-cyclic AMP-induced HAChT rise. As evidence that enhanced HAChT is associated with a direct action of cyclic AMP-dependent pathways on the cholinergic nerve terminals, addition of 8-bromocyclic AMP to isolated hippocampal synaptosomes induced an increase of HAChT that was prevented by H-89. Choline acetyltransferase activity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db-cyclic AMP addition. Our studies show that cyclic AMP-mimetic compounds appear to modulate the choline carrier by a dual mode: an early increase of the maximal velocity without a change of the number of HC-3 binding sites and a late rise of transport that is accompanied by an increase of HC-3 binding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.

Intracellular levels of cyclic AMP and cyclic GMP differentially modify platelet aggregate size in human platelets activated with epinephrine or ADP.

We investigated the effects on human platelet aggregation of several agents that increase either intracellular cyclic AMP or cyclic GMP, using a platelet aggregometer that allows quantification of the size and number of platelet aggregates. During the initial phase of aggregation induced by epinephrine and ADP, small aggregates consisting of < 100 cells predominated; large aggregates formed later. Prostaglandin I2 (PGI2), which increases intracellular cyclic AMP, suppressed the formation of small as well as large aggregates induced by epinephrine, with ID50 values of 10.7 +/- 2.8 and 3.8 +/- 0.5 nM, respectively. ADP-induced formation of small and large aggregates was also inhibited by PGI2, with similar ID50 values. Dibutyryl cyclic AMP (db cyclic AMP), a cell-permeant form of cyclic AMP, also inhibited small and large aggregate formation induced by epinephrine or ADP, with ID50 values of 420-560 microM for small aggregates and 139-166 microM for large aggregates, respectively. On the other hand, nitroprusside, which increases intracellular cyclic GMP, inhibited only the formation of large aggregates, with an ID50 value of 454 +/- 191 nM for epinephrine-induced activation and of 2.1 +/- 0.6 microM for ADP-induced activation. Nitroprusside at 1 mM did not affect the formation of small aggregates induced by epinephrine, whereas that of large aggregates was completely blocked at 10 microM. 8-Bromo cyclic GMP (8-br cyclic GMP) also inhibited only the formation of large aggregates, with ID50 values of 140-170 microM, but not that of small aggregates induced by epinephrine and ADP. Milrinone, which increases the intracellular level of both cyclic AMP and cyclic GMP, suppressed the formation of small and large aggregates induced by epinephrine and ADP. These findings suggest that cyclic AMP and cyclic GMP differentially modify the size of aggregates formed during epinephrine or ADP activation.

Enhancing effect of staurosporine on NO production in rat peritoneal macrophages via a protein kinase C-independent mechanism.

Staurosporine (3-100 nM), frequently used as a protein kinase C (PKC) inhibitor, increased accumulation of nitrite in the culture medium of rat peritoneal macrophages up to 6 times above the control level. Moreover, when used in combination with the stable analogue of cyclic AMP, dibutyrylcyclic AMP (db cyclic AMP; 0.1 mM), and/or a cytokine, tumour necrosis factor-alpha (TNF alpha; 100 u ml-1), staurosporine synergistically potentiated, up to 30 times, nitrite accumulation. On the other hand, the other PKC inhibitors, calphostin C and H-7 (10 nM-10 microM) were not effective under the same conditions. The staurosporine-induced nitrite accumulation, in both the presence and the absence of TNF alpha and/or db cyclic AMP was effectively inhibited by the protein synthesis inhibitor, cycloheximide, or by the nitric oxide (NO) synthesis inhibitor, NG-monomethyl-L-arginine (L-NMMA). Thus our data suggest that staurosporine may enhance NO production in macrophages via intracellular mechanisms unrelated to the PKC inhibition.

Potentiation of P1075-induced K+ channel opening by stimulation of adenylate cyclase in rat isolated aorta.

1. The effects of analogues and stimulators of cyclic AMP on the 86Rb+ efflux-stimulating and binding properties of P1075, an opener of ATP-dependent potassium channels, were studied in rat aortic rings. The increase in 86Rb+ efflux stimulated by P1075 was taken as a qualitative measure of K+ channel opening. 2. Forskolin, a direct activator of adenylate cyclase, isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and dibutyryl-cyclic AMP (db-cyclic AMP), a membrane permeant cyclic AMP-analogue, relaxed rat aortic rings contracted by noradrenaline with EC50 values of 0.06, 2 and 10 microM, respectively. 3. Forskolin, IBMX and db-cyclic AMP produced concentration-dependent increases of the 86Rb+ efflux induced by P1075 (50 nM) by up to twofold with EC50 values of about 0.1, 1.7 and 81 microM. At these concentrations the agents had little effect on the basal rate of 86Rb+ efflux. 4. The 86Rb+ efflux produced by P1075 in the presence of the cyclic AMP stimulators was inhibited by glibenclamide, a blocker of ATP-sensitive potassium channels. 5. IBMX (100 microM) induced a leftward shift of the concentration-86Rb+ efflux curve of P1075 without increasing the maximum. The enhancements of P1075-stimulated 86Rb+ efflux produced by combinations of forskolin and IBMX were either additive or less than additive. 6. The protein kinase A inhibitor, H-89, inhibited P1075-stimulated 86Rb+ efflux in the presence of IBMX significantly more than in the absence of IBMX, suggesting that the effect of increased cyclic AMP levels is mediated by protein kinase A. 7. At high concentrations, forskolin and IBMX slightly increased basal 86Rb+ efflux and inhibited the tracer efflux induced by P1075.8. Binding of [3H]-P1075 to rat aortic rings was either unaffected or inhibited by forskolin, IBMX and db-cyclic AMP.9. This study shows that moderate stimulation of the cyclic AMP system potentiates the K+ channel opening effect of P1075 by activation of protein kinase A. The fact that binding of [3H]-P1075 remains unchanged or is diminished favours the hypothesis that the K'channel openers activate ATP-dependent K+ channels by an indirect mechanism.

Functional PAF receptors in glia cells: Binding parameters and regulation of expression

Platelet activating factor is a unique phosphoglycerine which possesses a variety of biological functions exerting its biological effects via specific surface receptors. In the central nervous system, platelet activating factor has been suggested to play a role during injury especially in conditions of ischemia and trauma-induced neuronal damage. The specific cell populations expressing platelet activating factor receptor, however, have not been identified. In this study, the binding properties of platelet activating factor receptors in C6 glioma cells and primary cultures of astrocytes and oligodendrocytes were characterized by using the ligand [3H]WEB 2086. Early-passage glial cells which exhibit oligodendrocytic phenotype, expressed lower levels of [3H]WEB 2086 binding than either late-passage cells which exhibit astrocytic phenotypes or primary astroglia cells. No specific binding was observed in primary cultures of oligodendrocytes. The Bmax (136 ± 15.3 fmol/mg protein) and Kd (29 ± 3.2 nM) levels obtained for primary astroglia cells were similar to those described for other cell types. The expression of platelet activating factor receptor in early-passage glia cells was up-regulated by treatment with insulin which induces astrocytic differentiation. In contrast, db-cyclic AMP exerted an inhibitory effect on the level of platelet activating factor receptor in both early- and late-passage cells. The level of functional platelet activating factor receptor in C6 cells as measured by the ability of platelet activating factor to induce 45Ca2+ influx was increased in cells expressing astrocytic phenotypes and was decreased in db-cyclic AMP-treated cells. In accordance with lack of specific [3H]WEB 2086 binding, platelet activating factor did not induce a detectable response of Ca2+ influx in cultures of oligodendrocytes. This report provides the first direct demonstration of selective expression of functional platelet activating factor receptors and their properties in astroglia cells. The findings support the suggestion that platelet activating factor may play an important role as a mediator of injury and immune responses in the nervous system.

Role of cyclic AMP-induced Cl conductance in aqueous humour formation by the dog ciliary epithelium.

1. The effects of isoprenaline, a forskolin derivative NKH-477, and dibutyryl cyclic AMP (db cyclic AMP) on the membrane potential, conductance and cell volume of the dog non-pigmented ciliary epithelium (NPE) were investigated by intracellular potential recording, nystatin-perforated patch clamp technique and videomicroscopic cytometry. 2. The resting membrane potential of NPE was about -70 mV in physiological saline and was depolarized by isoprenaline in a dose-dependent manner with an ED50 of about 3 nM. This depolarization was competitively antagonized by the beta-adrenoceptor antagonist, timolol (pA2 = ca. 9) and almost completely blocked by the Cl transport blocker, DIDS. 3. In single dissociated NPE cells, 10 microM isoprenaline induced an inward current and caused a concomitant decrease in cell volume. The reversal potential measurement indicated that this inward current was carried mainly by Cl ion. DIDS (10 microM) abolished both the current and cell volume decrease. 4. NKH-477 (10 microM) or db cyclic AMP (1 mM) also induced an inward current together with a cell volume decrease, the properties of which were similar to those caused by isoprenaline. 5. These results suggest that beta-adrenoceptor stimulation in NPE leads to an increased rate of aqueous humour production by increasing Cl- efflux via an elevation of cyclic AMP and this effect is efficiently blocked by timolol.

Potentiation by adenosine of ATP‐evoked dopamine release via a pertussis toxin‐sensitive mechanism in rat phaeochromocytoma PC12 cells

1. The effects of adenosine on adenosine 5'-triphosphate (ATP)-evoked dopamine release from rat phaeochromocytoma PC12 cells was investigated to determine whether adenosine exerts a regulatory effect on the ATP-evoked response. Adenosine potentiated ATP (30 microM)-evoked dopamine release in a concentration-dependent manner over a concentration-range of 1 to 100 microM. Adenosine (100 microM) shifted the concentration-dependence of the ATP-evoked response to the left without affecting the maximal response. 2. Aminophylline, a non-selective adenosine receptor antagonist, and CP66713, a selective antagonist at the A2 subclass of adenosine receptors, abolished the adenosine-induced potentiation. Furthermore, 8-cyclopentyltheophylline, a selective antagonist at the adenosine A1 receptor partially inhibited the adenosine-evoked potentiation. CGS22492, a selective A2 receptor agonist, potentiated ATP-evoked dopamine release whereas N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, had no effect. 3. Pertussis toxin (PTX), a bacterial exotoxin which catalyzes the ADP-ribosylation of guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins), inhibited the adenosine-induced potentiation of dopamine release. Dibutyryl cyclic AMP (db cyclic AMP), an analogue of cyclic AMP, had no effect on the release on the ATP-evoked response. 4. Adenosine potentiated the ATP-evoked rise in intracellular Ca2+ concentration ([Ca]i) in PC12 cells. This potentiation was also observed with CGS 22492 but not with CHA. PTX completely inhibited the adenosine-induced potentiation of the rise in [Ca]i. 5. On the basis of these findings, we suggest that the adenosine-induced potentiation of ATP-evoked dopamine release was due to an increase in [Ca]i in the cells. Although the potentiation is most likely mediated by a subclass of A2 receptors, the subclass may be different from those previously reported since the potentiation was sensitive to PTX and was not reproduced by db cyclic AMP.

Induction of nitric oxide synthase in cultured vascular smooth muscle cells: the role of cyclic AMP.

1. Interleukin-1 beta (IL-1 beta) is a potent stimulant of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production in vascular smooth muscle (VSM) cells in culture. These studies investigate the role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in this process. 2. Dibutyryl cyclic AMP (db cyclic AMP, 0.1-1 mM), forskolin (1-10 microM) and the phosphodiesterase inhibitor, Ro 20-1724 (1-10 microM), all of which increase intracellular cyclic AMP, had no effect on NO production when added alone but markedly enhanced NO production by IL-1 beta-stimulated VSM cells in a dose-dependent manner. Consistent with a cyclic AMP-mediated action, isoprenaline (1-10 microM) increased NO production from IL-1 beta-stimulated cells. Dibutyryl cyclic GMP (db cyclic GMP) had no effect at concentrations up to 1 mM. 3. Pursuing these observations, iNOS protein levels were examined by Western blot analysis and iNOS mRNA levels were measured by reverse transcription and amplification of the resultant cDNA using the polymerase chain reaction. In addition to enhancing NO production, db cyclic AMP increased iNOS protein and mRNA above that produced by IL-1 beta alone. 4. These data demonstrate a major effect of cyclic AMP on cytokine-induced NOS activity in VSM cells, mediated at least in part by regulating synthesis of iNOS, and has implications for the pathogenesis and management of septic shock.

Cholera toxin-induced G sα down-regulation in neural tissue: studies on the pineal gland

Cholera toxin (CT) treatment (50 μg/ ml) was used to down regulate the α subunit of the stimulatory guanine nucleotide binding protein (Gsα) in pineal glands in organ culture, as has been seen in non-neural tissue. A 15 h treatment reduces G sα by ≈ 75% as measured using semi-quantitative Western blot technology. In contrast, this treatment does not alter the abundance of Gβ, G iα or G oα. This effect on G sα was still apparent following a 36-h washout period. The 48-h CT treatment increased cyclic AMP accumulation 10- to 17-fold but blocked the norepinephrine (NE)-induced increase in cyclic AMP accumulation, presumably reflecting the loss of G sα. This treatment did not, however, inhibit protein synthesis or stimulation of arylalkylamine N-acetyltransferase (NAT) activity produced by treatment with either DB-cyclic AMP ( N 6,2′-O-dibutyryl adenosine 3′,5′monophosphate) or 8 Br-cyclic AMP, stable cyclic AMP derivatives. This indicates that a 48-h CT treatment was not generally toxic. In contrast, this treatment blocked subsequent CT stimulation of NAT. The effects of CT treatment on the adrenergic stimulation of NAT was examined using treatments which selectively produced α- or β-adrenergic stimulation. α 1-Adrenergic activation of the pineal gland elevates [Ca 2+] i, which potentiates effects of cyclic AMP; in these studies the response to α-adrenergic activation was markedly increased in 48-h CT-treated glands, reflecting Ca 2+ potentiation of the effects of elevated levels of cyclic AMP. In contrast, the effects of the selective β-adrenergic agonist isoproterenol was reduced by ≈ 75%. These studies not only establish CT-induced G sα down-regulation as a new tool for the study of adrenergic signal transduction in the pineal gland, but indicate that this paradigm is probably useful in all neural tissue.

Effects of dibutyryl cyclic AMP on Na+,K(+)-ATPase activity and intracellular Na+ and K+ in primary cultures of astrocytes from DBA and C57 mice.

Effects of chronic treatment of dibutyryl cyclic AMP (db-cyclic AMP) on Na+,K(+)-ATPase activity in cell homogenates and intracellular Na+ and K+ contents [(Na+)i and (K+)i] were studied in primary cultures of astrocytes derived from cerebral cortex of neonatal audiogenic seizure-susceptible DBA and audiogenic seizure-resistant C57 mice. Na+,K(+)-ATPase activity in cell homogenates was greater and (Na+)i was less in DBA astrocytes than in C57 astrocytes. There was no difference in (K+)i between astrocytes from DBA and C57 mice. Addition of db-cyclic AMP to the medium from day 14 to day 21 in culture (final concentration 0.25 mM) increased Na+,K(+)-ATPase activity in cell homogenates and decreased (Na+)i, but had no significant effect on (K+)i in astrocytes from either DBA or C57 mice. Chronic treatment with db-cyclic AMP altered cell growth. Protein and DNA content of cultured astrocytes from both DBA and C57 mice was decreased. DNA was more affected than protein. Modifying K+ and Na+ concentration in medium altered Na+,K(+)-ATPase activity in cell homogenates as well as (Na+)i and (K+)i in cultured astrocytes of both DBA and C57 mice. Changes in (Na+)i and (K+)i at different K+ concentrations in medium paralleled those in Na+,K(+)-ATPase activity in cell homogenates. Results indicate that the ability to transport Na+ across the cell membrane and the response of Na+,K(+)-ATPase to db-cyclic AMP and to the changes in K+ in medium of cultured astrocytes from audiogenic seizure-susceptible DBA mice are sufficient.

Interferon-gamma increases cytoplasmic motility of alveolar macrophages via nitric oxide-dependent signaling pathways.

The effects of interferon-gamma (IFN-gamma) were investigated on cytoplasmic motility of alveolar macrophages (AM) from rat lungs in vitro. Cytoplasmic motility was examined by measuring remnant field strength from the cell surface of AM containing Fe3O4 particles, and the relaxation rate (lambda 0; min-1), which is related to cytoplasmic motility, was determined. IFN-gamma caused an increase in lambda 0 in a concentration-dependent fashion with the maximal effect at 1,000 U. Dibutyryl cyclic GMP (db cyclic GMP; 10(-3) M) mimicked IFN-gamma-induced effects, but db cyclic AMP (10(-3) M) decreased lambda 0. IFN-gamma (1,000 U)-induced increases in lambda 0 were concentration-dependently inhibited by NG-monomethyl-L-arginine (L-NMMA) with complete inhibition of a concentration of 10(-4) M and were also completely inhibited by either methylene blue (10(-5) M) or KT 5823 (10(-5) M), a specific inhibitor of protein kinase G. IFN-gamma (1,000 U) caused significant nitrite (NO2-) production from the control values of 0.2 +/- 0.1 to 10.0 +/- 0.2 microM/24 h per 10(6) cells (P < 0.001, n = 10), and this increase in NO2- production by IFN-gamma (1,000 U) was completely inhibited by L-NMMA (10(-4) M). IFN-gamma caused a concentration-dependent increase in a filamentous-actin (F-actin) content with the maximal effect at 1,000 U. db cyclic GMP (10(-3) M) mimicked IFN-gamma induced effects on F-actin formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory actions of cyclic AMP on neurogenic plasma extravasation in rat airways.

To determine whether neurogenic inflammation can be inhibited by cyclic AMP, which is suggested to have an inhibitory effect on neuropeptide release from airway sensory nerves, we examined plasma extravasation in the airways of anesthetized rats in vivo with Evans blue dye as a marker. Neurogenic inflammation was produced by antidromic electrical stimulation of the right vagus nerve (4 Hz, 1 ms, 4 V for 1 min). Electrical stimulation significantly increased leakage of dye in the trachea and right bronchus. Forskolin (from 0.01 to 100 pM/kg), dibutyryl cyclic AMP (db cyclic AMP; from 10 pM/kg to 100 nM/kg) and fenoterol (from 100 to 1 nM/kg) dose dependently inhibited the leakage of dye induced by electrical stimulation in the trachea and right bronchus. Substance P (1 microgram/kg) increased Evans blue dye extravasation in the same way as the leakage induced by electrical stimulation. Forskolin (from 0.1 to 1 pM/kg), db cyclic AMP (1 nM/kg) and fenoterol (10 nM/kg) failed to inhibit substance P-induced leakage, but showed significant inhibitory effects on the leakage induced by electrical stimulation in the trachea and right bronchus. However, further increases in the concentrations of forskolin, db cyclic AMP and fenoterol significantly inhibited substance P-induced leakage of dye in both tissues. These results suggest that cyclic AMP inhibits neurogenic plasma leakage by presynaptic inhibition of the release of neuropeptides from sensory nerves as well as by postsynaptic effects on the vascular endothelium.

Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities.

1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL-1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.

Depression of miniature endplate potential frequency by acetylcholine and its analogues in frog.

1. Acetylcholine (ACh), 7.5 x 10(-5) M, and carbachol, 5 x 10(-6) M (CCh) depressed the frequency of miniature endplate potentials (m.e.p.ps) in the frog (Rana temporaria) sartorius neuromuscular junction with active acetylcholinesterase to about 50-55% of the controls. 2. A similar depression was produced by the nicotinic agonists, nicotine, suberyldicholine and tetramethylammonium. 3. The muscarinic agonists, oxotremorine, methylfurmethide and methacholine were without effect on m.e.p.p. frequency. The muscarinic antagonist, atropine and the nicotinic antagonist, (+)-tubocurarine, had no effect on the depression of m.e.p.p. frequency evoked by CCh. 4. The ganglionic blockers, benzhexonium and IEM-1119, were also without effect on the CCh-evoked depression of m.e.p.p. frequency. 5. Pretreatment of muscles with anticholinesterases did not prevent the CCh-induced drop in m.e.p.p. frequency. 6. The effect of CCh was proportionally the same as in the controls in preparations where the m.e.p.p. frequency was changed by elevation of K+ and in the presence of theophylline, noradrenaline, dibutyryl adenosine 3':5'-cyclic monophosphate (db cyclic AMP) and db cyclic GMP. 7. An inhibitor of Na+,K(+)-ATPase, ouabain, 5 x 10(-5) mol l-1, prevented or reversed the depression of m.e.p.p. frequency by CCh. However, the depression was present in a nominally K(+)-free medium. Insulin and adrenaline, which are considered to be Na+,K(+)-ATPase activators, were without effect on depression of m.e.p.p. frequency. 8. The depression of m.e.p.p. frequency by 5 x 10(-6) M CCh was the same at temperatures between 5 and 30 degrees C with a Q10 near to 1.0. When threshold amounts of CCh were used (6 x 10-7 and 3 x 10-7 M), the depression was less at higher temperatures.9. The receptive structures responsible for the CCh (or ACh)-evoked depression of m.e.p.p. frequency differ pharmacologically from muscarinic, nicotinic ganglionic and neuromuscular junction ACh-receptors as well as from the synaptic cholinesterase, in contrast to previous reports (Duncan & Publicover, 1979).The low temperature-dependence points to the possibility that physical rather than biochemical processes are limiting in this presynaptic effect of cholinomimetics.