A simple andsensitive assay foradenosine 3':5'-cyclic monophos- phate(cAMP)hasbeendeveloped thatisbasedoncompetition forprotein binding ofthenucleotide, presumably toacAMP-dependent protein kinase. Thenucleotide-protein complex isadsorbed onacellulose ester filter. Assay conditions aresuchthatabinding constant approaching 10-9M\1 isobtained, and theassay isthussensitive to0.05-0.10 pmolofcAMP. Whileassays foradenosine 3':5'-cyclic monophosphate (cAMIP) areproliferat- ingalmost asrapidly asarenewlydiscovered actions ofthenucleotide, the tremendous current interest inthebiological role ofcAMPdictates theneedfora sensitive, accurate, andreadily performed procedure forestimation ofitscellular levels. Themethods currently availablel-8 donothavetheextreme sensitivity required bythelowtissue levels ofthecompound, orelsetheyarelaborious toperform, orboth. Thepresent study wasinitiated withthethought thatthebinding of(5H)- cAMP toacAMP-dependent protein kinase9-12 wouldformthebasis fora sensitive assay, ifasimple meanscould befound toisolate theprotein-nucleotide complex. Further impetus was-provided bythediscovery ofWalshetal.3 that aheat-stable protein, aninhibitor ofthecAMP-dependent protein kinase,14"15 increased theaffinity ofthecyclic nucleotide fortheenzyme. 'Aprotein kinase frommuscle waschosen forinvestigation because ofitsfavorable binding con- stant forcAMP.16Itwasreadily determined thatcAMP-dependent protein kinase andcAMP-binding activities frommuscle extracts could bequantitatively adsorbed oncellulose ester (Millipore) filters. Thebinding ofcAMPtospecific proteins fromEscherichia coli andtheadrenal cortex hasrecently beenstudied onMillipore filters.17'18 Thissimple meansforestimating cAMPbinding maybe utilized forassay ofunknownquantities ofthecyclic nucleotide indeproteinized tissue extracts. Aslittle as0.05-0.10 pmolofcAMP canbedetected; thus, less thanmilligram quantities ofmanytissues aresufficient forassay. Materials andMethods.cAMP-dependent protein kinase:Two purification schemes havebeenemployed-a moreextensive onetocharacterize theprotein andasimplified methodforroutine use.Procedures arepatterned after thoseof Walshetat.9 andMiyamoto etal."' Freshbovine muscle wasprepared asdescribed" through theammoniumsulfate precipitation step.Thisfraction, from250gofmuscle, wasapplied toacolumn ofDEAE-cellulose (Whatinan DE 11,1meq/g; 32X 2.6cm), previously equilibratcd with5mMIpotassium phosphate, pH7,andtile columni was

http://www.pnas.org/content/67/1/305.full.pdf

A Protein BindingAssayforAdenosine 3':5'-Cyclic Monophosphate

Caffeine is one of the most widely consumed substances found in beverages, and has demonstrated anticancer effects in several types of cancer. The present study aimed to examine the anticancer effects of caffeine on gastric cancer (GC) cells (MGC‑803 and SGC‑7901) in vitro, and to determine whether the apoptosis‑related caspase‑9/-3 pathway is associated with these effects. The sustained antiproliferative effects of caffeine on gastric cancer were also investigated. GC cell viability and proliferation were evaluated using cell counting and colony forming assays, following treatment with various concentrations of caffeine. Flow cytometry was performed to assess cell cycle dynamics and apoptosis. Western blot analysis was conducted to detect the activity of the caspase‑9/-3 pathway. The results indicated that caffeine treatment significantly suppressed GC cell growth and viability and induced apoptosis by activating the caspase‑9/-3 pathway. Furthermore, the anticancer effects of caffeine appeared to be sustained, as the caspase‑9/-3 pathway remained active following caffeine withdrawal. In conclusion, caffeine may function as a sustained anticancer agent by activating the caspase‑9/-3 pathway, which indicates that it may be useful as a therapeutic candidate in gastric cancer.

/pdf/caffeine-induces-sustained-apoptosis-of-human-gastric-cancer-1ei45e77u7.pdf

Caffeine induces sustained apoptosis of human gastric cancer cells by activating the caspase‑9/caspase‑3 signalling pathway.

Microglial-driven changes in synaptic plasticity: A possible role in major depressive disorder.

Evidence demonstrates that chronic caffeine exposure, primarily through consumption of coffee or tea, leads to increased alertness and anxiety. Preclinical and clinical studies showed that caffeine induced beneficial effects on mood and cognition. Other studies using molecular techniques have reported that caffeine exhibited neuroprotective effects in animal models by protecting dopaminergic neurons. Moreover, caffeine interacts with dopaminergic system, which leads to improvements in neurobehavioral measures in animal models of depression or attention deficit hyperactivity disorder (ADHD). Glutamatergic receptors have been found to be involved on the neurobiological effects of caffeine. Additionally, caffeine has been found to suppress the inhibitory (GABAergic) activity and modulate GABA receptors. Studies have also found that modulating these neurotransmitters leads to neurobehavioral effects. The linkage between the modulatory role of caffeine on neurotransmitters and neurobehavioral effects has not been fully discussed. The purpose of this review is to discuss in detail the role of neurotransmitters in the effects of caffeine on neurobehavioral disorders.

Caffeine induces neurobehavioral effects through modulating neurotransmitters.

Caffeine: well-known as psychotropic substance, but little as immunomodulator.

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors.

Caffeine alters glutamate–aspartate transporter function and expression in rat retina

The spontaneously hypertensive rat (SHR) is an excellent animal model that mimics the behavioral and neurochemical phenotype of attention-deficit/hyperactivity disorder (ADHD). Here, we characterized the striatal GABA transport of SHR and investigated whether caffeine, a non-selective antagonist of adenosine receptors, could influence GABAergic circuitry. For this purpose, ex vivo striatal slices of SHR and Wistar (control strain) on the 35th postnatal day were dissected and incubated with [3H]-GABA to quantify the basal levels of uptake and release. SHR exhibited a reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system. GAT-1 appears to be the primary transporter for [3H]-GABA uptake in SHR striatum, as GAT-1 selective blocker, NO-711, completely abolished it. We also verified that acute exposure of striatal slices to caffeine improved [3H]-GABA uptake and release in SHR, whereas Wistar rats were not affected. GABA-uptake increase and cAMP accumulation promoted by caffeine was reverted by A1R activation with N6-cyclohexyl adenosine (CHA). As expected, the pharmacological blockade of cAMP-PKA signaling by H-89 also prevented caffeine-mediated [3H]-GABA uptake increment. Interestingly, a single caffeine exposure did not affect GAT-1 or A1R protein density in SHR, which was not different from Wistar protein levels, suggesting that the GAT-1-dependent transport in SHR has a defective functional activity rather than lower protein expression. The current data support that caffeine regulates GAT-1 function and improves striatal GABA transport via A1R-cAMP-PKA signaling, specifically in SHR. These results reinforce that caffeine may have therapeutic use in disorders where the GABA transport system is impaired.

Danielle Dias Pinto Ferreira

Papers

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors.

Caffeine alters glutamate–aspartate transporter function and expression in rat retina

Caffeine Improves GABA Transport in the Striatum of Spontaneously Hypertensive Rats (SHR).