The terminal differentiation of malignant melanoma cells is known to be induced by activating cAMP signaling pathway with alpha-MSH or cAMP analogues. However, sustained activation of cAMP signaling system that induces the differentiation of melanoma cells, also induces the desensitization of the pathway at the receptor level. Nevertheless, the adaptation of adenylate cyclase (AC) expression by sustained activation of cAMP signaling system has not been clearly understood. This study was performed to examine whether the sustained activation of cAMP system induce changes in the expression AC isoforms as an adaptation mechanism. Treatment of B16/F10 murine melanoma cells with 100 mM forskolin for 6 days resulted in differentiation, melanin accumulation and increased expression of tyrosine hydroxylase mRNA. In the forskolin-treated melanoma cells, change in expression of various AC isoform at the transcription level was detected by reverse-transcription polymerase chain reaction (RT-PCR). Expression of AC isoform mRNA: ACI, III, VI, VII, and IX increased to the level of 196-392% of the control whereas the level of ACII was decreased by 30%. The cAMP concentration was increased both in basal and alpha-MSH stimulated cells, but the AC activity was decreased in the forskolin treated cells. Thus, these results suggest that sustained activation of cAMP system induces differential expression of AC isoforms, which results in increase of cAMP accumulation.
Adenylate Cyclase/*genetics ; Animal ; Cell Differentiation ; Cyclic AMP/*metabolism ; Forskolin/*pharmacology ; Isoenzymes/genetics ; Melanoma, Experimental/*enzymology/*pathology ; Mice ; Signal Transduction

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and was known to stimulate the release of growth factor in various cells. Recently, we reported the cellular localization of PACAP and its type I (PAC1 ) receptor in rat placenta during pregnancy. Placenta is a critical organ that synthesizes several growth factors and angiogenic factors for the fetal development and its own growth. However, there is little information regarding the cellular localization of PACAP and its receptor in human placenta at various gestations. The aim of the present study was to define the expression and distribution of PACAP and PAC1 receptor mRNAs in the human placenta during the pregnancy period. PACAP and PAC1 receptor mRNAs were expressed in stroma cells of stem villi and terminal villi. At the early stage, on 7 and 14 weeks, PACAP and PAC1 receptor genes were moderately expressed in stroma cells surrounding the blood vessels within stem villi. These genes were strongly expressed in stroma cells of stem villi and terminal villi on 24 and 38 weeks. The expression of these genes was increased as gestation advanced, and localized in the same areas. Localization of PACAP and PAC1 receptor demonstrate the evidence that PACAP may play an important role, as an autoregulator or pararegulator via its PAC1 receptor. In conclusion, our findings strongly suggest that PACAP may have a critical role in physiological function of the placenta for gestational maintenance and fetal growth.
Chorionic Villi/metabolism ; Female ; Gene Expression ; Humans ; Nerve Growth Factors/*biosynthesis ; Neuropeptides/*biosynthesis ; Neurotransmitter Agents/*biosynthesis ; Pituitary Adenylate Cyclase-Activating Polypeptide ; Placenta/*metabolism ; Pregnancy ; Pregnancy Trimester, First ; Pregnancy Trimester, Second ; RNA, Messenger ; Receptors, Cell Surface/*biosynthesis ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I

OBJECTIVETo investigate the expression changes and regulation of pituitary adenylate cyclase-activating polypeptide (PACAP) mRNA in corpus luteum during pregnancy.

METHODSPregnant rats' ovaries were collected at different time points. The techniques of RT-PCR and in situ hybridization were used to observe expression changes of PACAP mRNA in rat ovaries during pregnancy. To further explore the regulation mechanism of PACAP mRNA expression in corpus luteum, luteal cells were cultured in vitro. Immature (25 - 28 days old) female Sprague-Dawley rats were injected subcutaneously with 50IU pregnant mare serum gonadotrophin (PMSG), and 25IU human chorionic gonadotrophin (hCG) 48 h later, to induce follicular development and luteum formation. On day 6 after hCG administration (the day of hCG administration was the first day), the rats were killed by guillotine and the ovarian luteal cells were collected. After incubation for 24 h, luteal cells were administration with various factors for 24 h. And then expression changes of PACAP mRNA in luteal cells after administration with different factors were detected by RT-PCR, and radioimmunoassay was used to analyze progesterone levels.

RESULTSWith the development of pregnancy, the expression of PACAP mRNA increased gradually, reached the peak at pregnancy 19 d, and then decreased. Compared with control group, platelet activating factor (PAF), forskolin and PMA could obviously stimulate PACAP mRNA expression in luteal cells which were cultured with corresponding factors for 24 h. At the same time, progesterone levels in culture media were also elevated.

CONCLUSIONPACAP, acting as a local ovary regulator, was closely related to the maintenance of medium-term and late pregnancy. PAF could directly stimulate PACAP mRNA expression in luteal cells, and protein kinase C (PKC) and protein kinase A (PKA) signal pathways could both participate in this process.

Animals ; Cells, Cultured ; Corpus Luteum ; metabolism ; Female ; Pituitary Adenylate Cyclase-Activating Polypeptide ; genetics ; metabolism ; Platelet Activating Factor ; metabolism ; Pregnancy ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

The present study was aimed at examining the regulation of aquaporin (AQP)-2 water channels in the kidney in two-kidney, one clip (2K1C) hypertension. Rats were made 2K1C hypertensive for 6 weeks, and their expression of AQP2 channel proteins was determined in the clipped and contralateral kidneys. To examine the upstream affecting AQP2 channels, adenylyl cyclase activity was also determined. Along with the hypertension, in the clipped kidney, the abundance of AQP2 proteins was significantly decreased in the cortex, outer and inner medulla, while their trafficking remained unaltered. Concomitantly with the reversal of the blood pressure at 24 hours following removal of the clip, the AQP2 abundance also returned to the control level. The arginine vasopressin-evoked generation of cAMP was decreased in the clipped kidney, which again was reversed to the control level following removal of the clip. In contrast, the expression of AQP2 channels as well as the activity of adenylyl cyclase remained unaltered in the contralateral kidney. These results indicate an altered regulation of AQP2 water channels in the clipped kidney in 2K1C hypertension.
Adenylate Cyclase/metabolism ; Animal ; Aquaporins/*analysis ; Blood Pressure ; Cyclic AMP/biosynthesis ; Hypertension, Renovascular/*metabolism ; Kidney/*chemistry ; Male ; Rats ; Rats, Sprague-Dawley

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

Animals ; Cells, Cultured ; Female ; Granulosa Cells ; metabolism ; Pituitary Adenylate Cyclase-Activating Polypeptide ; genetics ; metabolism ; Platelet Activating Factor ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

AIMTo observe the protective role of pituitary adenylate cyclase activating polypeptide (PACAP) on hippocampal neuronal apoptosis induced by beta amyloid peptide in the culture.

METHODSHippocampal neurons were isolated from 1d old SD rat and neuronal survival and apoptosis were measured by MTT assay and DNA ladder.

RESULTS25 micromol/L Abeta could induce neuron apoptosis while co-treatment with PACAP could increase the survival of hippocampal neurons. The antagonist of PACAP receptor, P6-27, could reverse the effect of PACAP.

CONCLUSIONPACAP could protects cultured neurons from the neurotoxicity of Abeta through the activation of PACAP receptor and may have a bright use in treatment of neurodegenerative disease.

Amyloid beta-Peptides ; toxicity ; Animals ; Apoptosis ; drug effects ; Cells, Cultured ; Hippocampus ; cytology ; Neurons ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide ; metabolism

OBJECTIVETo investigate the differentially expressed proteins among chronic sinusitis, nasal polyps and normal nasal mucosa by means of proteomic technology, and select the candidate biomarkers of chronic sinusitis and nasal polyps.

METHODSProteins extracted from chronic sinusitis, nasal polyps and normal nasal mucosa were separated and the differentially expressed proteins were identified by series of proteomic tools, including immobilized pH4-7 gradient two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, modified coomassie brilliant blue staining, images scanning by the Image Scanner apparatus, PDQuest analysis software, peptide mass fingerprinting based on matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) by in-gel digestion extract, and Mascot searching in NCBInr and SWISS-PROT databases.

RESULTSThe 2-DE patterns with high resolution and reproducibility were obtained. The protein spots separated and visualized in chronic sinusitis, nasal polyps and normal nasal mucosa gel were 1020 +/- 40, 1112 +/- 10 and 1008 +/- 25, respectively. And the match rates were (93 +/- 2)%, (95 +/- 1)% [see text] (90 +/- 3)% respectively. Thirteen differentially expressed spots were found from chronic sinusitis, nasal polyps and normal nasal mucosa gel. We selected and recommend Keratin 8 and APOA1 proteins as candidate biomarkers of nasal polyps, and PLUNC protein, PACAP protein, NKEF-B and SOD as candidate biomarkers of chronic sinusitis.

CONCLUSIONSThe differentially expressed proteins among chronic sinusitis, nasal polyps and normal nasal mucosa can be efficiently and relatively reliably identified via the techniques of proteomics. These techniques will play a very important role in the researches for new objective indicators possibly employed in the future classifying, staging and prognosis.

Adolescent ; Adult ; Apolipoprotein A-I ; metabolism ; Female ; Glycoproteins ; metabolism ; Humans ; Keratin-8 ; metabolism ; Male ; Middle Aged ; Nasal Mucosa ; metabolism ; Nasal Polyps ; diagnosis ; metabolism ; Phosphoproteins ; metabolism ; Pituitary Adenylate Cyclase-Activating Polypeptide ; metabolism ; Proteomics ; Sinusitis ; diagnosis ; metabolism ; Young Adult

AIMIn order to study the effects of pituitary adenylate cyclase activating polypeptide(PACAP) on brain edema induced by ischemia in rats and its underlying receptor mechanism.

METHODSBrain ischemia model in rats was established by ligaturing four--vessels. The percentage ratio of wet over dry tissue weight, sodium and potassium contents of dry brain tissue were measured by weighing and enzymatic analysis methods.

RESULTSThe brain water contents significantly increased after rats exposed to 1 h of reperfusion following 30 - minute ischemia. Furthermore, sodium contents in brain tissue increased and potassium contents decreased following perfusion. Changes of brain water contents, sodium and potassium contents were relieved by lateral ventricular injection of PACAP in the concentration of 1 x 10(-9), 1 x 10(-10) or 1 x 10(-11) mol respectively before ischemia. The effect of PACAP could be blocked by MCAP6 - 38 (specific type I PACAP receptor antagonist) lateral ventricular injection prior to PACAP administration.

CONCLUSIONExogenous PACAP may act as a protective effect in brain edema induced by ischemia in rats, which is mediated by type I receptor.

Animals ; Brain ; metabolism ; physiopathology ; Brain Edema ; etiology ; metabolism ; prevention & control ; Brain Ischemia ; complications ; metabolism ; Male ; Neuropeptides ; metabolism ; Pituitary Adenylate Cyclase-Activating Polypeptide ; pharmacology ; Potassium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium ; 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