BMP-2 Is Involved in Scleral Remodeling in Myopia Development

The development of myopia is associated with scleral remodeling, but it is unclear which factors regulate this process. This study investigated bone morphogenetic protein-2 (BMP-2) expression in the sclera of guinea pigs with lens-induced myopia (LIM) and after recovery from myopia and evaluated the effect of BMP-2 on extracellular matrix (ECM) synthesis in human scleral fibroblasts (HSFs) cultured in vitro. Lens-induced myopia was brought about in two groups of guinea pigs (the lens-induced myopia and myopia recovery groups) by placing -4.00 D lenses on the right eye for three weeks. The left eye served as a contralateral control. In the recovery group, the lenses were removed after one week. The refractive power and axial length of the eyes were measured, and the BMP-2 expression levels in the sclera were measured. After three weeks, the lens-induced eyes acquired relative myopia in both groups of guinea pigs. Immunostaining of the eyeballs revealed significantly decreased BMP-2 expression in the posterior sclera of the myopic eyes compared to the contralateral eyes. One week after lens removal, BMP-2 expression recovered, and no differences were observed between the experimental and contralateral eyes in the recovery group. HSFs were cultured with BMP-2 or transforming growth factor-β1 (TGF-β1). Type I and type III collagen synthesis was significantly up-regulated following BMP-2 treatment in culture after one and two weeks, but the ratio of type III to type I collagen mRNA was not increased. Biosynthesis of glycosaminoglycan (GAG) and aggrecan was increased in HSFs treated with BMP-2. Some chondrogenesis-associated genes expression increased in HSFs treated with BMP-2. From this study, we concluded that BMP-2 is involved in scleral remodeling in the development and recovery of lens-induced myopia.     

大鼠垂体促性腺激素细胞内cAMP的改变对LHβ mRNA表达的影响

目的 分析大鼠LHβ mRNA表达的促性腺激素释放激素(GnRH)受体后信号转导机制.方法 将体外培养的大鼠腺垂体促性腺激素(GTH)细胞用cAMP的兴奋剂FSK或抑制剂SQ22536处理后,再用高频GnRH脉冲刺激,然后用实时荧光定量PCR法测定细胞LHβ mRNA的Ct值,并与空白组比较.结果 LHβ mRNA的Ct值随着GTH细胞cAMP含量的增高而显著降低,随着cAMP含量的降低而显著增高.结论 cAMP是高频GnRH脉冲刺激所引起的LHβ mRNA表达的受体后的信号转导途径.     

改变大鼠促性腺激素细胞内cAMP含量对卵泡刺激素分泌的影响

目的:分析大鼠卵泡刺激素(FSH)分泌的受体后信号转导机制。方法:将促性腺激素(GTH)细胞用毛喉素(FSK)或腺苷酸环化酶抑制剂SQ22536处理后,用促性腺激素释放激素脉冲刺激,再用酶联免疫吸附法检测其FSH分泌量,并与空白对照组比较。结果:FSK能显著提高GTH细胞中环磷酸腺苷(cAMP)含量,SQ22536能显著降低GTH细胞中的cAMP含量,FSK和SQ22536都不会影响GTH细胞的蛋白激酶C活性,GTH细胞cAMP含量的变化对FSH分泌的影响不显著。结论:cAMP-PKA(蛋白激酶A)不是FSHβ亚基分泌的受体后信号转导途径。     

Retinoscleral control of scleral remodelling in refractive development: a role for endogenous FGF-2?

AIMS: Studies in avian models of myopia have shown that refractive error development can be influenced by exogenously delivered fibroblast growth factor (FGF)-2. The present study sought to determine whether endogenous FGF-2 was associated with retinoscleral signalling or scleral remodelling during changes in refractive error in a mammalian model of myopia. METHODS: Myopia was induced in tree shrews over a 5-day period. One group of animals was then allowed 3 days of recovery from the induced myopia. Endogenous levels of FGF-2 were measured in scleral and retinal homogenates using ELISA. Real-time PCR was used to investigate scleral FGF-2 and FGF receptor (FGFR)-1 mRNA expression. RESULTS: No difference in FGF-2 content was found in posterior scleral or retinal extracts of myopic eyes (scleral -4+/-9%, retinal +23+/-17%) or recovering eyes (scleral -10+/-18%, retinal +1+/-13%), when compared with contralateral control eyes. In addition, no significant changes were found in scleral FGF-2 mRNA expression in myopic or recovering eyes (+106+/-56% and +14+/-12% respectively, P=0.21). However, FGF-2 concentration was significantly higher in anterior, relative to posterior, scleral regions in all animals (1602+/-105 vs 1030+/-50pg/mg respectively P<0.001). Expression of scleral FGFR-1 mRNA was upregulated in myopic eyes (+186+/-32%, P=0.01) but returned to control eye levels during recovery (+63+/-20%). CONCLUSIONS: The findings indicate that alterations in endogenous retinal or scleral FGF-2 levels are not associated with changes in scleral remodelling in this mammalian model of myopia. However, the reversible changes found in FGFR-1 expression in the sclera of myopic eyes mean that an indirect role for FGF-2 in the control of scleral remodelling is implicated. The anteroposterior difference found in scleral FGF-2 concentration indicates a role for this cytokine in the control of normal scleral growth and development and, presumably, eye size.     

The Adenylyl Cyclase Inhibitor MDL-12,330A Potentiates Insulin Secretion via Blockade of Voltage-Dependent K+ Channels in Pancreatic Beta Cells

Objective

Adenylyl cyclases (ACs) play important role in regulating pancreatic beta cell growth, survival and secretion through the synthesis of cyclic AMP (cAMP). MDL-12,330A and SQ 22536 are two AC inhibitors used widely to establish the role of ACs. The goal of this study was to examine the effects of MDL-12,330A and SQ 22536 on insulin secretion and underlying mechanisms.

Methods

Patch-clamp recording, Ca²⁺ fluorescence imaging and radioimmunoassay were used to measure outward K⁺ currents, action potentials (APs), intracellular Ca²⁺ ([Ca²⁺]_i) and insulin secretion from rat pancreatic beta cells.

Results

MDL-12,330A (10 µmol/l) potentiated insulin secretion to 1.7 times of control in the presence of 8.3 mmol/l glucose, while SQ 22536 did not show significant effect on insulin secretion. MDL-12,330A prolonged AP durations (APDs) by inhibiting voltage-dependent K⁺ (K_V) channels, leading to an increase in [Ca²⁺]_i levels. It appeared that these effects induced by MDL-12,330A did not result from AC inhibition, since SQ 22536 did not show such effects. Furthermore, inhibition of the downstream effectors of AC/cAMP signaling by PKA inhibitor H89 and Epac inhibitor ESI-09, did not affect K_V channels and insulin secretion.

Conclusion

The putative AC inhibitor MDL-12,330A enhances [Ca²⁺]_i and insulin secretion via inhibition of K_V channels rather than AC antagonism in beta cells, suggesting that the non-specific effects is needed to be considered for the right interpretation of the experimental results using this agent in the analyses of the role of AC in cell function.     

Isoform-specific changes in scleral transforming growth factor-beta expression and the regulation of collagen synthesis during myopia progression

The development of high myopia is associated with altered scleral extracellular matrix biochemistry. Previous studies highlight the importance of collagen turnover in this process, yet it is unclear which factors control scleral remodeling. This study used a mammalian model of myopia to investigate the capacity of TGF (transforming growth factor)-beta1, -beta2, and -beta3 to influence scleral remodeling in myopia. RT-PCR confirmed the presence of all mammalian TGF-beta isoforms in scleral tissue and scleral fibroblasts. Myopia was experimentally induced via monocular deprivation of pattern vision, and animals were allocated to two groups depending on the duration of treatment (1 or 5 days). Down-regulation of each isoform was apparent after only 1 day of myopia development (TGF-beta1, -32%; TGF-beta2, -27%; TGF-beta3, -42%). Whereas the decrease in TGF-beta1 and -beta3 expression was relatively constant between the two time points, differential down-regulation of TGF-beta2 was found between days 1 (-27%) and 5 (-50%). In vitro experiments, using primary scleral fibroblasts, demonstrated the capacity of all isoforms to increase collagen production in a dose-dependent manner. Changes in TGF-beta levels, which mimicked those during myopia induction, caused an approximately 15% reduction in collagen synthesis, which is qualitatively similar to those previously reported in vivo. These data represent the first demonstration of TGF-beta3 expression in the sclera and implicate all three TGF-beta isoforms in the control of scleral remodeling during myopia development. In addition, the early alterations in TGF-beta expression levels may reflect a role for these cytokines in mediating the retinoscleral signal that controls myopic eye growth.     

The effects of inhibitors of adenylate cyclase and phosphodiesterase on D. discoideum aggregation.

The action of two adenine analogues on the aggregation of D. discoideum amebae was examined. SQ22536 and SQ20009 are inhibitors of adenylate cyclase and phosphodiesterase, respectively, in higher eukaryotes. Both compounds are shown here to inhibit the differentiation of cells to aggregation-competence. SQ22536-treated cells exhibited normal accumulation of their adenylate cyclase activity as measured in cell lysates but the amebae did not synthesize cAMP. The ability of this drug to compete for cAMP surface-binding sites, and the observation that the effects of the drug could be reversed by imposed pulses of cAMP, suggest that SQ22536 functions as a cAMP antagonist. The effects of SQ20009 on cell differentiation did not appear to be mediated by an inhibition of phosphodiesterase activity or cAMP binding to the cell surface. Amebae were arrested at a very early stage in development and remained unresponsive to external cAMP.     

Myopia Development in Tree Shrew Is Associated with Chronic Inflammatory Reactions

In this study, we aimed to investigate whether chronic retinal inflammation is involved in the pathogenesis of form-deprivation myopia (FDM) using tree shrews as an animal model. Twenty-one tree shrews were randomly divided into 7-day/14-day FDM (FDM7/FDM14) groups and their corresponding 7-day/14-day control groups. Refraction and axial length were measured. To determine the effects of form deprivation on inflammation, we used real-time polymerase chain reaction (PCR) and immunohistochemistry to assess the expression levels of several proinflammatory cytokines. At day 0, the eyes in the FDM and control groups were hyperopic. However, after 7 and 14 days of form deprivation, the refractive error of the eyes in the FDM7 and FDM14 groups shifted from +6.6 ± 0.3 diopters (D) to +4.0 ± 0.5 D and from +6.4 ± 0.3 D to +5.0 ± 0.3 D, respectively. The levels of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and nuclear factor κB were increased in the FDM eyes, compared with those in the control eyes. The increase in matrix metalloproteinase-2 expression was greater in the FDM eyes than in the contralateral and control eyes, whereas collagen type I expression was downregulated. In conclusion, chronic inflammation may play a crucial pathogenic role in form-deprivation myopia in tree shrews.     

Scleral Cross-Linking Using Riboflavin UVA Irradiation for the Prevention of Myopia Progression in a Guinea Pig Model: Blocked Axial Extension and Altered Scleral Microstructure

PurposeTo develop methods of collagen cross-linking (CXL) in the sclera for the treatment of progressive myopia and to investigate the biomechanical and histological changes that occur in as a result.MethodsTwenty 14-day-old guinea pigs were divided into 3 groups: the cross-linking group (CL, n = 8), non cross-linking group (NCL, n = 8), and control group (n = 4). The scleras of the right eyes of the guinea pigs in the CL group were surgically exposed and riboflavin was dropped onto the irradiation zone for 20 seconds prior to ultraviolet-A (UVA) irradiation. The same procedure was conducted on the NCL group but without UVA irradiation. No procedure was conducted on the control group. The right eyes of the guinea pigs in the CL and NCL groups were then fitted with -10.00DS optics for six weeks. Retinoscopy and the axial lengths (AXL) were measured at baseline, and at the second, fourth and sixth weeks post-treatment in all three groups. All animal subjects were euthanized after the sixth week and then biomechanical and histopathological examinations of the scleras were conducted.ResultsThe mean AXL of the NCL group was longer than both the control and CL groups at six weeks (P = 0.001). The mean refractive error in the NCL group was statistically significantly more negative than both the control and the CL groups at six weeks (P = 0.001). The scleral collagen fiber arrangements of the CL and control groups were denser and more regularly distributed than the NCL group. Ultimate stress of the sclera was lowest in the NCL group, followed by the CL then the control group (P<0.05). Ultimate strain (%) of the sclera was lowest in the CL group followed by the NCL and then the control group (P<0.05).ConclusionOur study demonstrates that scleral CXL using riboflavin UVA irradiation effectively prevents the progression of myopia by increasing scleral biomechanical strength in a guinea pig model.     

Wnt Signaling in Form Deprivation Myopia of the Mice Retina

Background

The canonical Wnt signaling pathway plays important roles in cellular proliferation and differentiation, axonal outgrowth, cellular maintenance in retinas. Here we test the hypothesis that elements of the Wnt signaling pathway are involved in the regulation of eye growth and prevention of myopia, in the mouse form-deprivation myopia model.

Methodology/Principal Findings

(1) One hundred twenty-five C57BL/6 mice were randomly distributed into form-deprivation myopia and control groups. Form-deprivation myopia (FDM) was induced by suturing the right eyelid, while the control group received no treatment. After 1, 2, and 4 weeks of treatment, eyes were assessed in vivo by cycloplegic retinoscopic refraction and axial length measurement by photography or A-scan ultrasonography. Levels of retinal Wnt2b, Fzd5 and β-catenin mRNA and protein were evaluated using RT-PCR and western blotting, respectively. (2) Another 96 mice were divided into three groups: control, drugs-only, and drugs+FDM (by diffuser). Experimentally treated eyes in the last two groups received intravitreal injections of vehicle or the proteins, DKK-1 (Wnt-pathway antagonist) or Norrin (Wnt-pathway agonist), once every three days, for 4 injections total. Axial length and retinoscopic refraction were measured on the 14th day of form deprivation.Following form-deprivation for 1, 2, and 4 weeks, FDM eyes had a relatively myopic refractive error, compared with contralateral eyes. There were no significant differences in refractive error between right and left eye in control group. The amounts of Wnt2b, Fzd5 and β-catenin mRNA and protein were significantly greater in form-deprived myopia eyes than in control eyes.DKK-1 (antagonist) reduced the myopic shift in refractive error and increase in axial elongation, whereas Norrin had the opposite effect in FDM eyes.

Conclusions/Significance

Our studies provide the first evidence that the Wnt2b signaling pathway may play a role in the development and progression of form-deprivation myopia, in a mammalian model.     

The Sonic Hedgehog Signaling Pathway Induces Myopic Development by Activating Matrix Metalloproteinase (MMP)-2 in Guinea Pigs

Purpose

To investigate whether the Sonic hedgehog (Shh) signaling induces myopic development by increasing the expression of matrix metalloproteinase (MMP)-2 in guinea pigs.

Methods

A translucent diffuser was glued onto the right eye to induce form-deprivation myopia (FDM) in 10 guinea pigs. Four guinea pigs were served as a control group. The other 100 guinea pigs were subdivided into 5 groups (20 per group) and received a 10 µl intravitreal injection every 2 days for 4 times. Two groups were injected with 20 or 50 µg/ml Shh amino-terminal peptide (Shh-N) into the right eye and 0.1% bovine serum albumin into the other. FDM was induced in the right eyes of the three cyclopamine-treated groups and both eyes were injected with 50, 100, or 200 µg/ml cyclopamine. Retinoscopic refraction and eye dimensions were assessed on Day 14 of treatment. MMP-2 protein expression was determined in both scleras by western blotting.

Results

Both concentrations of Shh-N stimulated myopic development and axial growth as compared with control eyes. Myopia and axial elongation were significantly greater in the 50 µg/ml than in the 20 µg/ml Shh-N group (P<0.001 and P = 0.0019, respectively). All three doses of cyclopamine significantly attenuated myopic development compared with the FDM group (P<0.0001). Cyclopamine at 100 or 200 µg/ml significantly reduced axial elongation compared with the FDM group (P = 0.044 and P = 0.001, respectively). FDM-induced myopia and axial elongation were significantly greater in the 50 µg/ml than in the 200 µg/ml cyclopamine group (P<0.0001 and P = 0.008, respectively). MMP-2 expression was significantly greater in Shh-N–treated eyes than in the control eyes, and was lower in the cyclopamine plus FDM groups than in the FDM group.

Conclusions

The Shh signaling pathway induces myopic development by activating MMP-2 in guinea pigs.     

The cyclic AMP-dependent protein kinase A pathway is involved in progesterone effects on calcitonin secretion from TT cells

It is well known that gonadal steroid hormones influence the level of plasma calcitonin (CT), but the mechanism by which progesterone affects CT secretion is not clear. Immortalized TT cells are a reliable model system for studying the endocrine function of human parafollicular cells. In the present study, the effects of progesterone on CT secretion were examined in TT cells. TT cells were incubated in medium containing vehicle (DMSO), progesterone or BSA-progesterone for 60 or 150 min, and then the levels of CT in the medium, progesterone receptors, cAMP accumulation and CT mRNA expression were measured. To study the correlation between progesterone effects and the cAMP-dependent protein kinase A (PKA) pathway, cell lysates or cells in 24-well plates were treated with either vehicle or progesterone plus RU486, SQ22536, KT5720, or 3-isobutyl-1-methylxanthine. Then, adenylyl cyclase and protein kinase A (PKA) activities were measured in the cell lysates, and the CT levels were measured in the medium from the 24-well plate. The activated cAMP response element binding protein (P-CREB) was also measured by immunofluorescence. Administration of 1 microM progesterone or 500 nM BSA-progesterone increased the secretion of CT by 381% and 100%, respectively. Progesterone receptors A and B were downregulated by progesterone treatment. The cAMP concentration, adenylyl cyclase and PKA activity, CT mRNA expression, and nuclear P-CREB concentrations all showed an increase after progesterone treatment. RU486, SQ22536 and KT5720 inhibited the progesterone-stimulated effects. These results suggest that a cAMP-dependent PKA pathway is involved in progesterone-stimulated effects on CT secretion from TT cells.     

AQP1 gene expression is upregulated by arginine vasopressin and cyclic AMP agonists in trophoblast cells

Aquaporins (AQPs) are water channels that regulate water flow in many tissues. As AQP1 is a candidate to regulate placental fluid exchange, we sought to investigate the effect of arginine vasopressin (AVP) and cAMP agonists on AQP1 gene expression in first trimester-derived extravillous cytotrophoblasts (HTR-8/Svneo) and two highly proliferative carcinoma trophoblast-like cell lines but with a number of functional features of the syncytiotrophoblast namely; JAR and JEG-3 cells. Our data demonstrated that AVP (0.1 nM) significantly increased the expression of AQP1 mRNA at 10 h in HTR-8/SVneo and JEG-3 cells (P<0.05). Both SP-cAMP, a membrane-permeable and phosphodiesterase resistant cAMP, and forskolin, an adenylate cyclase stimulator significantly increased AQP1 mRNA expression in all cell lines after 2 h in a dose-dependent manner (P<0.05) with a parallel increase in protein expression. In the time course study, 5 microM of either SP-cAMP or forskolin significantly stimulated AQP1 mRNA expression after 2 h in HTR-8/SVneo cells and after 10 h in JAR and JEG-3 cells. AQP1 protein expression was highest after 20 h in both HTR-8/SVneo and JEG-3 cells (P<0.05). AVP-stimulated cAMP elevation was blocked in the presence of 9-(tetrahydro-2'-furyl) adenine (SQ22536) (100 microM), a cell-permeable adenylate cyclase inhibitor (P<0.05). These results indicate that in trophoblasts-like cells AQP1 gene expression is upregulated by both AVP and cAMP agonists. Furthermore, our data demonstrate that a cAMP-dependent pathway is responsible for the AVP effect on AQP1. Thus, modulation of AQP1 expression by maternal hormones may regulate invasion and fetal-placental-amnion water homeostasis during gestation.     

Role of autocrine stimulation on the effects of cyclic AMP on protein and lipid phosphorylation in collagen-activated and thrombin-activated platelets.

We compared several responses in thrombin-stimulated and collagen (type I)-stimulated platelets with and without forskolin and inhibitors of autocrine stimulation (IAS: an ADP-removing system of creatine phosphate/creatine phosphokinase, Arg-Gly-Asp-Ser peptide to prevent fibrinogen/fibronectin binding to GPIIb/IIIa, SQ 29.548 as a thromboxane A2 receptor antagonist, cyproheptadine as a serotonin receptor antagonist, BN 52021 as a platelet-activating factor receptor antagonist). The pattern of tyrosine-phosphorylated proteins, the phosphorylation of lipids in the polyphosphoinositide cycle and phosphorylation of pleckstrin (P47) were studied as markers for signal-transducing responses, exposure of CD62 (P-selectin) and CD63 (Glycoprotein 53), as well as secretion of ADP + ATP and beta-N-acetyl-glycosaminidase were studied as final activation responses. Clear differences between thrombin-stimulated and collagen-stimulated platelets were observed. First, practically all protein-tyrosine phosphorylation induced by thrombin was inhibited by IAS, while a partial inhibition was observed for collagen; the phosphorylation due to collagen alone was apparently stimulated by elevation of cAMP. Secondly, the other responses to thrombin were inhibited by increased levels of cAMP, independent of autocrine stimulation. In contrast, only the autocrine part of the collagen-induced responses was inhibited by elevation of cAMP. Thus, the inhibition by elevated cAMP seen in collagen-stimulated platelets seems to be due to removal of the G-protein-mediated activation from secreted autocrine stimulators either by IAS or forskolin. The remaining activity is a pure collagen effect which is not affected by elevated levels of cAMP.     

Regulation of ethanol-sensitive EAAT2 expression through adenosine A1 receptor in astrocytes

Adenosine-regulated glutamate signaling in astrocytes is implicated in many neurological and neuropsychiatric disorders. In this study, we examined whether adenosine A1 receptor regulates EAAT2 expression in astrocytes using pharmacological agents and siRNAs. We found that adenosine A1 receptor-specific antagonist DPCPX or PSB36 decreased EAAT2 expression in a dose-dependent manner. Consistently, knockdown of A1 receptor in astrocytes decreased EAAT2 mRNA expression while overexpression of A1 receptor upregulated EAAT2 expression and function. Since A1 receptor activation is mainly coupled to inhibitory G-proteins and inhibits the activity of adenylate cyclase, we investigated the effect of forskolin, which activates adenylate cyclase activity, on EAAT2 mRNA levels. Interestingly, we found that forskolin reduced EAAT2 expression in dose- and time-dependent manners. In contrast, adenylate cyclase inhibitor SQ22536 increased EAAT2 expression in dose- and time-dependent manners. In addition, forskolin blocked ethanol-induced EAAT2 upregulation. Taken together, these results suggest that A1 receptor-mediated signaling regulates EAAT2 expression in astrocytes.     

Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles

Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.     

Adenosine A1 receptor-mediated inhibition of myocardial norepinephrine release involves neither phospholipase C nor protein kinase C but does involve adenylyl cyclase

Stimulation of adenosine A1 receptors in the heart exerts cardioprotective effects by inhibiting norepinephrine (NE) release from sympathetic nerve endings. The intraneuronal signal transduction triggered by presynaptic adenosine A1 receptors is still not completely understood. The objective of the present study was to determine whether phospholipase C (PLC), protein kinase C (PKC), and adenylyl cyclase (AC) are involved in the adenosine A1 receptor-mediated inhibition of endogenous (stimulation-induced) NE release in isolated Langendorff-perfused rat hearts as an approach to elucidate their role in the cardiovascular system. Activation of adenosine A1-receptors with 2-chloro-N6-cyclopentyladenosine (CCPA) decreased cardiac NE release by approximately 40%. Inhibition of PLC with 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U 73122) as well as inhibition of PKC with 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide (GF 109203X) slightly but significantly decreased NE release; however, the suppressive effect of CCPA on NE release was not modulated by U 73122 or GF 109203X. Blockade of AC with 9-(tetrahydro-2'-furyl)adenine (SQ 22536) reversed the inhibitory effect of CCPA on sympathetic neurotransmitter release irrespective of whether PKC was pharmacologically activated by phorbol 12-myristate 13-acetate or was not activated, indicating a PKC-independent but AC-dependent mechanism. Direct stimulation of AC with forskolin increased NE release by approximately 20%; an effect that was antagonized by either CCPA or SQ 22536. These data suggest that the adenosine A1 receptor-mediated inhibition of NE release does not involve PLC or PKC but does involve AC.