MTT analysis and intracellular calcium measurement by using confocal laser scanning microscopy were used to study the possible mechanism of protective effect of pituitary adenylate cyclase activating polypeptide 27 (PACAP27) from beta amyloid protein (Abeta)-induced neurotoxicity. The results showed that treatment with PACAP (less than 0.1 micromol/L) increased the survival and reproductive ability of neuro-2a cells and protected the neuro-2a cells from being injured by Abeta. The protective effect of PACAP27 was reversed by the competitive PACAP receptor antagonist PACAP6-27. An increase in intracellular calcium was observed when the cells were challenged with Abeta and PACAP. But the calcium increase induced by Abeta kept stable for a long time while PACAP caused a transient rise in intracellular calcium. The intracellular calcium increase induced by Abeta was blocked by pretreatment with PACAP for 10 min. It is suggested that the neuroprotective effect of PACAP against neuronal damage induced by Abeta may result from its role in inhibiting the sustained rise in intracellular calcium.
Amyloid beta-Peptides ; antagonists & inhibitors ; toxicity ; Calcium ; metabolism ; Calcium Channels ; metabolism ; Cell Line, Tumor ; Humans ; Neuroblastoma ; pathology ; Neuroprotective Agents ; pharmacology ; Pituitary Adenylate Cyclase-Activating Polypeptide ; pharmacology

It is now well established that several G protein- coupled receptors can signal without agonist stimulation (constitutive receptors). Inverse agonists have been shown to inhibit the activity of such constitutive G protein-coupled receptor signaling. Agonist activation of the Gi/o-coupled peripheral cannabinoid receptor CB2 normally inhibits adenylyl cyclase type V and stimulates adenylyl cyclase type II. Using transfected COS cells, we show here that application of SR144528, an inverse agonist of CB2, leads to a reverse action (stimulation of adenylyl cyclase V and inhibition of adenylyl cyclase II). This inverse agonism of SR144528 is dependent on the temperature, as well as on the concentration of the cDNA of CB2 transfected. Pertussis toxin blocked the regulation of adenylyl cyclase activity by SR 144528.
Adenylate Cyclase/antagonists&inhibitors/genetics/metabolism ; Animals ; Binding, Competitive ; Bornanes/metabolism/*pharmacology ; COS Cells ; Cannabinoids/metabolism ; Cercopithecus aethiops ; Isoenzymes/antagonists&inhibitors/genetics/metabolism ; Pyrazoles/metabolism/*pharmacology ; Rats ; *Receptor, Cannabinoid, CB2 ; Receptors, Cannabinoid ; Receptors, Drug/agonists/*antagonists&inhibitors/genetics/metabolism ; Signal Transduction/drug effects/physiology ; Transfection

According to our previous studies together with others, GnRH, a hypothalamic decapeptide, has been known to be a major regulator for LH release and its subunit biosynthesis in anterior pituitary gonadotropes. But the precise mechanisms by which GnRH exerts stimulatory effects on LH release and its subunit biosynthesis have not been clearly understood. In the present study we examined the effect of GnRH on protein kinase C (PKC) activity and intracellular cAMP content in cultured anterior pituitary cells of rat to clarify whether PKC or cAMP are involved in GnRH action. Moreover, we examined the effects of staurosporine (ST), a PKC inhibitor and 2',3'-dideoxyadenosine (2',3'-DDA), an adenylate cyclase inhibitor, on LH release and steady state LH beta subunit mRNA levels in cultured anterior pituitary cells of rat. PKC activity was rapidly increased within 30 min after GnRH treatment whereas intracellular cAMP level was elevated 18 h after GnRH treatment. ST significantly inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner, showing an half maximal response at 50 nM ST. 2',3'-DDA inhibited GnRH-induced LH release and LH beta subunit mRNA levels in a dose-dependent manner in pituitary cells. From these results, it is suggested that GnRH stimulates LH beta subunit mRNA level as well as LH release in anterior pituitary cells and this GnRH action might be mediated by PKC activation and cAMP stimulation.
Adenylate Cyclase/*antagonists & inhibitors ; Alkaloids/*pharmacology ; Animal ; Cells, Cultured ; Cyclic AMP/metabolism ; Dideoxyadenosine/*pharmacology ; Female ; Gonadorelin/*pharmacology ; Luteinizing Hormone/*biosynthesis/*metabolism ; Pituitary Gland, Anterior/*drug effects/metabolism ; Protein Kinase C/*antagonists & inhibitors/metabolism ; Rats ; Rats, Sprague-Dawley ; Staurosporine ; Support, Non-U.S. Gov't

OBJECTIVE: To explore the expression and significance of vasoactive intestinal peptide and Pituitary adenylate cyclase activiting polypeptide (VIP/PACAP) of nasal mucosa in rats with allergic rhinitis (AR), and the function of botulinum toxin-A(BTX-A) to inhibit the expression of VIP/PACAP in AR. METHOD: Thirty Sprague-Dawley rats were randomly divided into 3 groups, which were the AR group, the intervention group, and the control group. In the AR group, ovalbumin was used to sensitize healthy rats. In the intervention group, BTX-A was dripped into the nasal cavity of AR rats 7 times. In the control group, only physiological saline was used to drip into the nasal cavity of AR rats. Changes of the rats' behavior were observed. ELISA were used to detected the concentration variation of serum IFN-γ and IL-4. Histopathology and immunohistochemistry were employed to observe morphology in the rats' nasal mucosal and the expression of VIP/PACAP. Statistical analysis was also made. RESULT: (1)The typical symptoms marks of nasal scratching, sneezing, nasal blockage and rhinorrhea of AR group (7.5 ± 0.50) were higher than intervention group (1 ± 0.27) and control group (0.8 ± 0.31). (2) Comparing to intervention group and control group, the serm IFN-γ of the AR group obvious reduced (P < 0.05), the serm IL-4 of the AR group obvious rose (P < 0.01), and the serm Th1/Th2 (IFN-γ/IL-4) of the AR group obvious reduced (P < 0.01). (3) Comparing to intervention group and control group, the cilium loss, inflammatory cells infiltration, and inflammatory cells exudation of nasal mucosa in AR group were more obviously (P < 0.01), and the intervention group of the 3 indexes was obviously than control group. (4) The expression of VIP in the rats' nasal mucosa of the AR group (13.27 ± 2.74) were more intense than intervention group (5.21 ± 2.18) and control group (3.56 ± 5.30) (P < 0.01), and the expression of PACAP in the rats' nasal mucosa of the AR group (20.97 ± 2.14) were more intense than intervention group (6.33 ± 3.04) and control group (4.63 ± 1.25) (P < 0.01). (5) In all the 3 groups, there was positive correlation between expression of negative in VIP/PACAP and Thl/Th2 cell infiltration(r were respectively -0.340 and -0.223, P < 0.05). CONCLUSION The VIP/PACAP in the rats' nasal mucosa may play an important role in pathogenesis of AR, and BTX-A could improve the symptoms of AR through inhibition of the expression of VIP/ PACAP.
Animals ; Botulinum Toxins, Type A ; pharmacology ; Disease Models, Animal ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Nasal Mucosa ; drug effects ; metabolism ; Ovalbumin ; Paranasal Sinuses ; Pituitary Adenylate Cyclase-Activating Polypeptide ; antagonists & inhibitors ; metabolism ; Rats ; Rats, Sprague-Dawley ; Rhinitis, Allergic ; drug therapy ; Vasoactive Intestinal Peptide ; antagonists & inhibitors ; metabolism

Using site-directed mutagenesis technique, I have replaced serine 285 and serine 292 with the alanine, and assessed the binding of agonist and signaling such as the inhibition of adenylyl cyclase activity.I have found that serine 292 has an important role in the signal transduction of cannabinoid agonists, HU-210 and CP55940, but not in that of aminoalkylindoles derivatives WIN55,212-2. All mutants express well in protein level determined by western blot using monoclonal antibody HA 11 as compared with the wild type receptor.Interestingly, binding affinity of S285A and S292A mutants with classical cannabinoid agonist HU-243 was somewhat decreased. In signaling assay, the inhibition of adenylyl cyclase by HU-210, CP55940 and WIN55, 212-2 is the same order in both wild type receptor and S285A mutant receptor. However, S292A have been shown that the inhibition curves of adenylyl cyclase activity moved to the right by HU-210 and CP55940, but those of adenylyl cyclase activity did not by aminoalkylindole WIN55,212-2, which is indicating that this residue is closely related to the binding site with HU-210 and CP55940. In addition, serine 292 might take more important role in CB2 receptor and G-protein signaling than serine 285.
Adenylate Cyclase/*metabolism ; Animals ; Binding, Competitive ; Blotting, Western ; COS Cells ; Cannabinoids/metabolism ; Cercopithecus aethiops ; Cyclohexanols/metabolism ; Excitatory Amino Acid Antagonists/metabolism ; Mutagenesis, Site-Directed ; Protein Conformation ; Protein Structure, Tertiary ; Receptors, Cannabinoid ; Receptors, Drug/genetics/metabolism/*physiology ; Serine/metabolism/*physiology ; Signal Transduction/physiology ; Tetrahydrocannabinol/*analogs&derivatives/metabolism ; Transfection

VPAC2 is a co-receptor of pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) and mediates multiple bio-functions. In order to construct the CHO line expressing VPAC2 stably, pcDNA-VPAC2 was used to transfect CHO cells. The positive clones were selected by G418 and the clone VPAC2-CHO with high sensitivity to PACAP38 was picked out by its ability to promoting the concentration of cAMP. RT-PCR, Western blot and Immunofluorescenece assay were used to identify the express of VPACS. Binding competition with VPAC2 agonist and the bioactivity of mediating the ligand to promote the concentration of cAMP showed that VPAC2 was expressed effectively in VPAC2-CHO. The results of Scatchard analysis revealed that VAPC2-CHO expressed a receptor density of (1.1 +/- 0.2) pmol/mg protein, respectively, with Kd values of (0.55 +/- 0.10) nmol/L for PACAP38 used as a tracer. The construction of CHO cells expressing VPAC2 specially and functionally lays a foundation not only for the further research on the characters and functions of VPAC2 but also for the screening and characterization of novel agonists of antagonists for VPAC2.
Animals ; Binding, Competitive ; CHO Cells ; Cell Membrane ; drug effects ; metabolism ; Cricetinae ; Cricetulus ; Cyclic AMP ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Iodine Radioisotopes ; chemistry ; Pituitary Adenylate Cyclase-Activating Polypeptide ; chemistry ; metabolism ; pharmacology ; Receptors, Vasoactive Intestinal Peptide, Type II ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 micrometer) and bell-shaped. The concentrations of ATP, ATP-gammaS, ADP, and ADP-beta S that showed maximal IL-10 release were 100, 10, 100, and 100 micrometer respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP) = dATP > 2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), alpha,beta-methylene ATP (alpha,beta-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca2+ release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down- regulated by either MRS2179 (a P2Y1 antagonist) or 5'-AMPS (a P2Y11 antagonist), indicating that both the P2Y1 and P2Y11 receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-gammaS (100 micrometer) was restored by TNP-ATP (an antagonist of the P2X1, P2X3, and P2X4 receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y12 receptor antagonist) or pertusis toxin (PTX; a Gi protein inhibitor), indicating that the P2X1, P2X3, P2X4 receptor group, or the P2Y12 receptor, negatively modulate the P2Y11 receptor or the P2Y1 receptor, respectively.
Adenosine Diphosphate/analogs & derivatives/pharmacology ; Adenosine Triphosphate/analogs & derivatives/*pharmacology ; Adenylate Cyclase/antagonists & inhibitors ; Animals ; Calcium/metabolism ; Chelating Agents/pharmacology ; Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors ; Enzyme Inhibitors/pharmacology ; Extracellular Space/drug effects/*metabolism ; Gene Expression Regulation/drug effects ; Interleukin-10/*biosynthesis ; Microglia/*drug effects/enzymology/*metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor Cross-Talk/*drug effects ; Receptors, Purinergic P2/agonists/antagonists & inhibitors/genetics/*metabolism ; Thionucleotides/pharmacology