GLI2 induces PDGFRB expression and modulates cancer stem cell properties of gastric cancer.

OBJECTIVE: In this study, we aimed to investigate the downstream effector of GLI2 in gastric cancer (GC) and their regulative effect on cancer stem cell (CSC) properties of GC. MATERIALS AND METHODS: Bioinformatic data mining was performed in TCGA-Stomach Adenocarcinoma (STAD), as well as in Kaplan-Meier plotter. Moderate-differentiated GC cell line SGC-7901 and poor-differentiated GC cell line MKN-45 were used as in-vitro model to investigate the regulative effect of GLI2 on PDGFRB expression. MKN-45 cells were further used to explore the effect of GLI2 shRNA or PDGFRB shRNA on CSC properties of the cells. RESULTS: Bioinformatic results showed that GLI2 is usually upregulated in GC tissues than in normal tissues, and high GLI2 expression is associated with unfavorable first progression free survival (PFS) and also worse overall survival (OS) in patients with GC. PDGFRB is co-upregulated with GLI2 in GC and its promoter region contains a putative GLI2 binding site. The results of dual luciferase assay confirmed this binding site. Enforced GLI2 expression elevated PDGFRB expression at both mRNA and protein level. GLI2 or PDGFRB knockdown showed similar effect on reducing spheroid colony formation and on reducing the expression of CSC related genes, including CD44, Nanog, and Oct4 in MKN-45 cells. CONCLUSIONS: High GLI2 or PDGFRB expression is associated with unfavorable survival in GC patients. GLI2 can induce PDGFRB expression in GC cells via directly binding to its promoter. In addition, the GLI2-PDGFRB axis might be an important signaling pathway modulating CSC properties of GC cells.

Mediating effects of social support and self-concept on depressive symptoms in adults with spinal cord injury.

STUDY DESIGN: Cross-sectional, correlational design. OBJECTIVES: To examine the effects of individual demographics, activities of daily living, social support, and self-concept on depressive symptoms in people with spinal cord injury (SCI). SETTING: A convenience sample of 135 adults with SCI was recruited from medical and rehabilitation centres in Taiwan. METHODS: Face-to-face, structured interviews were employed to collect information. Study questionnaires included a demographic sheet, the Barthel scale, the modified Social Support Inventory, the Huang self-concept scale and the Beck Depression Inventory. Data were analysed by structural equation modelling (SEM). RESULTS: The average age of the participants was 43.3 years (±11.98), the mean duration of injury was 114 months (±93.78), and most were males. Emotional support (r=-0.173, P<0.05) and appraisal support (r=-0.261, P<0.01) were negatively correlated with depressive symptoms. The best fitted SEM model included individual demographics and physical function, social support and self-concept as significant predictors of depressive symptoms, with self-concept acting as a mediator in this relationship. Participants' characteristics and social support both contributed substantial indirect effects on depressive symptoms via self-concept. Self-concept also mediated the relationship between education, income, physical functioning and participants' depressive symptoms. CONCLUSION: For this sample, the more negative that individuals perceived themselves, the more likely they were to report worsening depressive symptoms. The more social support that individuals have, the more likely they were to report less depressive symptoms. Further longitudinal research will help clarify the direction of these relationships.

Mediating effects on health-related quality of life in adults with osteoporosis: a structural equation modeling.

UNLABELLED: Participants with physical limitation and high degree of pain had poor mental and physical health-related quality of life. In addition, the more support and exercise that the participants had, the more likely they were to report better health-related quality of life. INTRODUCTION: Osteoporosis is a public health threat worldwide. The aim of this study is to examine the effects of individual demographics, disease characteristics, and social support on health-related quality of life (HrQoL) of adults with osteoporosis. Most psychosocial studies focused on the relationships but not the specific construct of social support on HrQoL. METHODS: In a correlational design, face-to-face, structured interviews were employed to collect information. Study questionnaires included a demographic sheet, the modified Social Support Inventory, and the Short-Form 36 scales on a convenience sample of 161 individuals recruited from four outpatient centers. Using the structural equation modeling approach, all relationships among factors, mediators, and HrQoL were analyzed. RESULTS: The mean duration of osteoporosis was longer than 5 years. Participants who exercised more than three times per week had greater HrQoL than individuals who exercised less frequently. Participants with physical limitation and high degree of pain had poor mental and physical HrQoL. The more support that the participants perceived, the more likely they were to report better HrQoL. The best fitted structural equation modeling (SEM) model included individual demographics and physical function, and social support as significant predictors on HrQoL, with informational support and physical function acting as mediators in those relationships. Moreover, this structural model explained 35, 42, and 40 % of the variance on activity of daily living (ADL), physical, and mental health-related quality of life. CONCLUSIONS: The more informational support that individuals have, the more likely they were to report better HrQoL. Individuals with osteoporosis who have lower pain and more exercise are considered having better HrQoL. Further longitudinal research will help clarify the direction of these relationships.

Effect of germination temperature on characteristics of phytase production from barley.

The effects of germination temperature on the growth of barley seedlings for phytase production were studied at 15, 20 and 25 degrees C for 6-10 days. The growth rate of the barley seedlings was increased as the germination temperature was increased. The initial rate of total protein production was closely coupled to that of the barley growth, and the rate of total protein production tended to increase as the germination temperature was increased. SDS-PAGE analysis of total protein from the barley seedlings showed time-dependent appearance and disappearance of protein bands. Although no significant phytase activity was detected at zero time of germination, a significant increase in phytase activity up to 7.9-fold occurred during the first several days of germination then decreased. Phosphate production (viz. phytate degradation) in the barley seedlings occurred rapidly at the beginning of germination. However, the rate of production continued to decrease with further germination. A time lag of about 1-2 days between the rate of total protein production and that of phytase production was observed. Unlike the extent of total protein production, that of phytase production was similar irrespective of germination temperature. Partial purification of a crude enzyme extract by hydrophobic interaction chromatography resulted in two phytase fractions (PI and PII). Zymogram analysis demonstrated that PI had two bands with molecular masses of about 66 and 123 kDa while PII had one band corresponding to a molecular mass of about 96 kDa. The optimal temperature for PI was found to be 55 degrees C, while it was 50 degrees C for PII. The enzyme fraction PI had a pH optimum at 6.0, whereas the optimum pH for PII was found to be 5.0. Addition of 0.1% (v/v) Tween 80 was found to increase enzyme activity significantly (i.e., 167% for PI and 137% for PII). Phytate in cereals including barley, rice, corn and soybean degraded effectively by the treatment of the barley phytases.

N-glycosylation and residues Asn805 and Asn890 are involved in the functional properties of type VI adenylyl cyclase.

In this study, we demonstrate that type VI adenylyl cyclase (ACVI) is glycosylated in vivo. Treating HEK293 cells expressing ACVI with tunicamycin to block the addition of N-linked oligosaccharide or removing the N-linked oligosaccharide by in vitro peptidyl-N-glycosidase F digestion reduced the molecular mass of ACVI. Furthermore, tunicamycin treatment suppressed the forskolin-stimulated activity of ACVI. Mutation of either one or both potential N-glycosylation sites (Asn(805) and Asn(890), located on extracellular loops 5 and 6, respectively) also reduced the molecular mass of ACVI. Therefore, ACVI was glycosylated at both Asn(805) and Asn(890). Confocal analysis indicated that glycosylation was not required for the delivery of ACVI to the cell surface. Although no significant alterations in K(m) values for ATP or sensitivity to divalent cations were detected, the glycosylation-deficient ACVI mutant N805Q/N890Q-ACVI exhibited much lower forskolin-, Mn(2+)-, and Mg(2+)-stimulated cyclase activities than did wild-type ACVI. By contrast, the Galpha(s)-stimulated cyclase activities of wild-type ACVI and N805Q/N890Q-ACVI were indistinguishable. Furthermore, compared with wild-type ACVI, N805Q/N890Q-ACVI was less sensitive to inhibition mediated by dopamine D2 receptors or by protein kinase C. Collectively, glycosylation of ACVI not only affected its catalytic activity in an activator-dependent manner, but also altered its ability to be regulated by a Galpha(i) protein-coupled receptor or by protein kinase C.

Relaxation and music to reduce postsurgical pain.

AIMS: We investigated the effects of relaxation, music, and the combination of relaxation and music on postoperative pain, across and between two days and two activities (ambulation and rest) and across ambulation each day. This secondary analysis of a randomized controlled trial was conducted from 1995 to 1997. BACKGROUND: After surgery, patients do not always receive sufficient relief from opioids and may have undesired side-effects. More complete relief (10-30%) was found recently with adjuvant interventions of relaxation, music, and their combination. Comparison of effects between days and treatments have not been examined longitudinally. METHODS: With a repeated measures design, abdominal surgery patients (n = 468) in five US hospitals were assigned randomly to one of four groups; relaxation, music, their combination, and control. With institutional approval and written informed consent, subjects were interviewed and taught interventions preoperatively. Postoperative testing was at ambulation and rest on days 1 and 2 using visual analogue (VAS) sensation and distress of pain scales. RESULTS: Multivariate analysis indicated that although pain decreased by day 2, interventions were not different between days and activities. They were effective for pain across ambulation on each day, across ambulation and across rest over both days (all P < 0.001), and had similar effects by day and by activity. CONCLUSION: Nurses can safely recommend any of these interventions for pain on both postoperative days and at both ambulation and rest.

The clinical specialist in community health nursing: a solution for the 21st century.

The clinical specialist (CS) in community health nursing (CHN) addresses health problems in populations and communities in ways that are different from and complimentary to strategies used by practitioners of individualized patient care. Four programs based on comprehensive assessments, systematic program planning, deliberate implementation, and both formative and summative evaluation are presented. The programs were developed by graduate students in the CHN clinical specialization major at the Frances Payne Bolton School of Nursing. With expert faculty guidance and a competency-driven program of study, strategies were implemented to promote the health of community-dwelling well elderly people, home health care nurses who provide service to patients with respiratory problems, and high school students in a private religious school. Experienced parish nurses also benefited from a program designed to enhance their skills in theological reflection. This article also describes the curricular design developed to educate this clinician for the 21st century. Lastly, continued attention to the dynamic criteria of the American Nurses Association Standards of Community Health Nursing Practice is recommended as crucial to the further development of this clinical specialization.

In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams.

This study investigated the in vitro degradation of porous poly(DL-lactic-co-glycolic acid) (PLGA) foams during a 20-week period in pH 7.4 phosphate-buffered saline (PBS) at 37 degrees C and their in vivo degradation following implantation in rat mesentery for up to 8 weeks. Three types of PLGA 85 : 15 and three types of 50 : 50 foams were fabricated using a solvent-casting, particulate-leaching technique. The two types had initial salt weight fraction of 80 and 90%, and a salt particle size of 106-150 microm, while the third type had 90% initial weight fraction of salt in the size range 0-53 microm. The porosities of the resulting foams were 0.82, 0.89, and 0.85 for PLGA 85 : 15, and 0.73, 0.87, and 0.84 for PLGA 50 : 50 foams, respectively. The corresponding median pore diameters were 30, 50, and 17 microm for PLGA 85: 15, and 19, 17, and 17 microm for PLGA 50 : 50. The in vitro and in vivo degradation kinetics of PLGA 85: 15 foams were independent of pore morphology with insignificant variation in foam weight, thickness, pore distribution, compressive creep behavior, and morphology during degradation. The in vitro foam half-lives based on the weight average molecular weight were 11.1 +/- 1.8 (80%, 106-150 microm), 12.0 +/- 2.0 (90%, 106-150 microm), and 11.6 +/- 1.3 (90%, 0-53 microm) weeks, similar to the corresponding values of 9.4 +/- 2.2, 14.3 +/- 1.5, and 13.7 +/- 3.3 weeks for in vivo degradation. In contrast, all PLGA 50 : 50 foams exhibited significant change in foam weight, water absorption, and pore distribution after 6-8 weeks of incubation with PBS. The in vitro foam half-lives were 3.3 +/- 0.3 (80%, 106-150 microm), 3.0 +/- 0.3 (90%, 106-150 microm), and 3.2 +/- 0.1 (90%, 0-53 microm) weeks, and the corresponding in vivo half-lives were 1.9 micro 0.1, 2.2 +/- 0.2, and 2.4 +/- 0.2 weeks. The significantly shorter half-lives of PLGA 50: 50 compared to 85: 15 foams indicated their faster degradation both in vitro and in vivo. In addition, PLGA 50: 50 foams exhibited significantly faster degradation in vivo as compared to in vitro conditions due to an autocatalytic effect of the accumulated acidic degradation products in the medium surrounding the implants. These results suggest that the polymer composition and environmental conditions have significant effects on the degradation rate of porous PLGA foams.

In vitro degradation of porous poly(L-lactic acid) foams.

This study investigated the in vitro degradation of porous poly(L-lactic acid) (PLLA) foams during a 46-week period in pH 7.4 phosphate-buffered saline at 37 degrees C. Four types of PLLA foams were fabricated using a solvent-casting, particulate-leaching technique. The three types had initial salt weight fraction of 70, 80, and 90%, and a salt particle size of 106-150 microm, while the fourth type had 90% initial weight fraction of salt in the size range 0-53 microm. The porosities of the resulting foams were 0.67, 0.79, 0.91, and 0.84, respectively. The corresponding median pore diameters were 33, 52, 91, and 34 microm. The macroscopic degradation of PLLA foams was independent of pore morphology with insignificant variation in foam weight, thickness, pore distribution, compressive creep behavior, and morphology during degradation. However, decrease in melting temperature and slight increase in crystallinity were observed at the end of degradation. The foam half-lives based on the weight average molecular weight were 11.6+/-0.7 (70%, 106-150 microm), 15.8+/-1.2 (80%, 106-150 microm), 21.5+/-1.5 (90%, 106-150 microm), and 43.0+/-2.7 (90%, 0-53 microm) weeks. The thicker pore walls of foams prepared with 70 or 80% salt weight fraction as compared to those with 90% salt weight fraction contributed to an autocatalytic effect resulting in faster foam degradation. Also, the increased pore surface/volume ratio of foams prepared with salt in the range 0-53 microm enhanced the release of degradation products thus diminishing the autocatalytic effect and resulting in slower foam degradation compared to those with salt in the range 106-150 microm. Formation and release of crystalline PLLA particulates occurred for foams fabricated with 90% salt weight fraction at early stages of degradation. These results suggest that the degradation rate of porous foams can be engineered by varying the pore wall thickness and pore surface/volume ratio.

Metformin treatment enhances insulin-stimulated glucose transport in skeletal muscle of Sprague-Dawley rats.

Although the glucose-lowering properties of metformin are well-established, its effects on glucose metabolism in skeletal muscle have not been clearly defined. We tested the effects of metformin in young adult male Sprague-Dawley rats, which have a documented reduced response to insulin in skeletal muscle. Rats were treated with metformin for 20 days (320 mg/kg/day) in the drinking water. During this period, metformin completely prevented the increase in food intake and decreased adiposity by 30%. Metformin also reduced insulin secretion by 37% following an intra-peritoneal injection of glucose. Finally, metformin enhanced transport of [3H]-2-deoxyglucose in isolated strips of soleus muscle. Metformin substantially increased insulin-stimulated transport, while having no effect on basal transport. In control rats, a maximal concentration of insulin stimulated transport 77% above basal. In metformin-treated rats, insulin stimulated transport 206% above basal. We conclude that in the Sprague-Dawley rat model, metformin causes a significant increase in insulin-responsiveness.

The 5' untranslated regions of the rat A2A adenosine receptor gene function as negative translational regulators.

The rat A2A adenosine receptor (A2A-R) gene contains two promoters, P1 and P2, which produce transcript 1 and transcript 2, respectively. These transcripts differ in the lengths of their 5' untranslated regions (5'UTR1: 514 bp, initiated from P1; 5'UTR2: 221 bp, initiated from P2) but encode the same protein. In the present study, we demonstrate that transcript 2 is present in various tissues at different levels, whereas transcript 1 is found only in the striatum. In the striatum, the level of transcript 2 is approximately 300-fold higher than that of transcript 1. The 5'UTR of both transcripts suppresses the expression of A2A-R and a firefly luciferase reporter gene at the translational level; this suppression is not observed after mutational inactivation of an "out-of-frame" upstream AUG codon. Translational suppression by the 5'UTR was also confirmed in cells using a bicistronic strategy. Collectively, these data suggest that P2 is the major promoter of the rat A2A-R gene. The 5'UTR of the rat A2A-R gene exerts an inhibitory effect on translation by an upstream open reading frame. Because the 5'UTR of the A2A-R gene possesses strong interspecies homology, translational suppression may be a general mechanism by which the expression of the A2A-R gene is regulated.

The N terminus domain of type VI adenylyl cyclase mediates its inhibition by protein kinase C.

Previous results from our laboratory have shown that phosphorylation of type VI adenylyl cyclase (ACVI) by protein kinase C (PKC) caused suppression of adenylyl cyclase activity. In the present study, we investigated the role of the N terminus cytosolic domain of ACVI in this PKC-mediated inhibition of ACVI. Removal of amino acids 1 to 86 of ACVI or mutation of Ser(10) (a potential PKC phosphorylation site) into alanine significantly relieved the PKC-mediated inhibition and markedly reduced the PKC-evoked protein phosphorylation. PKC also effectively phosphorylated a recombinant N terminus cytosolic domain (amino acids 1-160) protein of ACVI and a synthetic peptide representing Ser(10). In addition, the amino acids 1 to 86 truncated mutant exhibited kinetic properties similar to those of the wild type. Taken together, these data demonstrate that the highly variable N terminus cytoplasmic domain of ACVI is a regulatory domain with a critical role in PKC-mediated suppression, which is a hallmark of this adenylyl cyclase isozyme. In addition, Ser(10) was found to serve as an acceptor for the PKC-mediated phosphorylating transfer of ACVI.

Modulation of dopamine transporter activity by nicotinic acetylcholine receptors and membrane depolarization in rat pheochromocytoma PC12 cells.

To elucidate the regulation of the rat dopamine transporter (rDAT), we established several PC12 variants overexpressing the rDAT. Treating these cells with a nicotinic agonist (1,1-dimethyl-4-phenylpiperazinium iodide, 30 microM) depolarized the plasma membrane potential from -31 +/- 2 to 43 +/- 5 mV and inhibited rDAT activity significantly in a calcium- and protein kinase C-independent manner. Membrane depolarization by a high external K+ concentration or two K+ channel blockers (tetraethylammonium hydroxide and BaCl2) also resulted in a marked inhibition of rDAT activity. Such inhibition of dopamine uptake is due to a reduction in Vmax, with no marked effect on the Km for dopamine. The potency of cocaine in inhibiting dopamine uptake was not significantly altered, whereas that of amphetamine was slightly enhanced by membrane depolarization. Removing extracellular Ca2+ or blocking the voltage-sensitive L-type calcium channels using nifedipine did not exert any significant effect on the inhibition of rDAT activity by depolarization. These data confirm that calcium influx on depolarization is not required for inhibition of the rDAT. Collectively, our data suggest that rDAT activity can be altered by a neurotransmitter that modulates the membrane potential, thus suggesting an exquisite mechanism for the fine-tuning of dopamine levels in the synapse.

Expression of type VI adenylyl cyclase in the central nervous system: implication for a potential regulator of multiple signals in different neurotransmitter systems.

The aim of this study was to investigate the distribution of a calcium-inhibitable adenylyl cyclase type VI (type VI AC) in the central nervous system using an antiserum directed against the N-terminus of type VI AC. Our results indicate that type VI AC immunoreactivity is generally expressed in many brain regions with different levels of intensity. Most interestingly, the majority of the detected type VI AC immunoreactivity is present in cells of neuronal phenotype. Double immunostaining of type VI AC and markers of various neurotransmitter systems suggest that type VI AC might participate in regulation of the classical neurotransmitter systems and therefore appeared to play a very important role in the central nervous system.

Activation of phosphodiesterase IV during desensitization of the A2A adenosine receptor-mediated cyclic AMP response in rat pheochromocytoma (PC12) cells.

Prolonged activation of an A2A adenosine receptor significantly inhibits the cellular response to subsequent stimulation (A2A desensitization). We have reported previously that activation of phosphodiesterase (PDE) contributes to A2A desensitization in PC12 cells. In the present study, we show that a type IV PDE (PDE4)-selective inhibitor (Ro 20-1724) effectively blocks the increase in PDE activity in desensitized cells. Thus, PDE4 appears to be the PDE specifically activated during A2A desensitization in PC12 cells. Prolonged treatment of PC12 cells with an A2A-selective agonist (CGS21680) leads to increased PDE4 activity in a dose-dependent manner, which can be blocked by an A2A-selective antagonist [8-(3-chlorostyryl)caffeine]. Using two PDE4 antibodies, we were able to demonstrate that the levels of two PDE4-immunoreactive bands (72 and 79 kDa) were increased significantly during A2A desensitization. Prolonged treatment with forskolin to elevate intracellular cyclic AMP contents also resulted in increased PDE4 activity. In addition, activation of PDE4 activity during A2A desensitization could be blocked by a protein kinase A (PKA)-selective inhibitor (H89) and was not observed in a PKA-deficient PC12 cell line (A123). Taken together, activation of PDE4 via a cyclic AMP/PKA-dependent pathway plays a critical role in dampening the signal of the A2A receptor.

Protein kinase C inhibits adenylyl cyclase type VI activity during desensitization of the A2a-adenosine receptor-mediated cAMP response.

We have previously reported that phosphorylation of adenylyl cyclase type VI (AC6) may result in the suppression of adenylyl cyclase activity during desensitization of the A2a-adenosine receptor-mediated cAMP response (A2a desensitization) in rat pheochromocytoma PC12 cells. In the present study, we demonstrate that protein kinase C (PKC) is responsible for the phosphorylation and inhibition of AC6 during A2a desensitization. Inhibition of PKC by several independent methods markedly blocked the suppression of AC6 during A2a desensitization. Purified PKC from rat brain directly phosphorylated and inhibited recombinant AC6 expressed in Sf21 cells. Substantially lower AC6 activities were also observed in PC12 cells overexpressing PKCdelta or PKCepsilon. Stimulation of A2a-R in PC12 cells under the same conditions as those required for A2a desensitization resulted in an increase in Ca2+-independent PKC activity. Most importantly, exogenous PKC did not further suppress AC6 activity in A2a-desensitized membranes. In vitro PKC phosphorylation of AC6 isolated from A2a-desensitized cells was also profoundly lower than that from control cells, suggesting a specific role for PKC in regulating AC6 during A2a desensitization in PC12 cells. Taken together, our data demonstrate that a calcium-independent, novel PKC inhibits AC6 activity during A2a desensitization in PC12 cells. Independent regulation of AC6 by calcium-independent PKC and by Ca2+ provides an exquisite mechanism for integrating signaling pathways to fine-tune cAMP synthesis.

Circadian rhythm in the Ca(2+)-inhibitable adenylyl activity of the rat striatum.

In the present study, we demonstrate that the Ca(2+)-inhibitable adenylyl cyclase (AC) activity in the striatum exhibits a daily oscillation with a peak occurring around 10:00 h. A circadian fluctuation of the AC activity evoked by an A2a adenosine-selective agonist was also observed. Intrastriatal injection of an A2a-selective adenosine agonist or antagonist during the interval in which the Ca(2+)-inhibitable AC activity was at its peak resulted in a more significant alteration of locomotor activity than those observed at a later interval. The marked circadian variation in the Ca(2+)-inhibitable AC activity in the striatum appears to cause a circadian fluctuation in the action of at least one neuromodulator.

Characterization of the rat A2a adenosine receptor gene.

To understand the molecular basis for the regulation of rat A2a adenosine receptor (A2a-R) expression, we have characterized the rat A2a-R gene and defined its promoter regions. Through a combination of restriction mapping and sequence analysis, we have demonstrated that the rat A2a-R gene is composed of two exons interrupted by a 7.2-kb intron. Primer extension and RNase protection on RNA isolated from PC12 cells suggested that the A2a-R gene encoded two clusters of alternative transcripts. The most upstream transcription start site was designated as +1. The sequence of the proximal 1.5 kb of 5'-flanking region demonstrated no potential TATA box, CCAAT box, or initiator element in the appropriate location. Varying lengths of 5'-flanking regions were inserted into a transient expression vector (pGL2-basic), which contained bacterial luciferase as the reporter gene, to determine its promoter region(s) in PC12 cells, CHOP cells, and C6 cells. Consistent with two clusters of transcription start sites, two independent functional promoter regions (designated P1, -67/-1; and P2, +272/+304) for the rat A2a-R gene were identified. Although both promoters are in use in PC12 cells, only P2 is active in CHOP cells, indicating possible cell line-specific usage of these two promoters.

Regulation of adenylyl cyclase type VI activity during desensitization of the A2a adenosine receptor-mediated cyclic AMP response: role for protein phosphatase 2A.

We reported earlier that inhibition of adenylyl cyclase activity is a mechanism involved in desensitization of the A2a adenosine receptor-mediated cAMP response (A2a desensitization) in rat pheochromocytoma PC-12 cells. Here, we investigated the molecular mechanism that modulates adenylyl cyclase activity during A2a desensitization. Reversible inhibition of forskolin-evoked adenylyl cyclase activity in desensitized cells occurred after incubation with an A2a-selective adenosine agonist (CGS21680). However, when okadaic acid (a relatively protein phosphatase 2A-specific phosphatase inhibitor) was added after agonist removal, adenylyl cyclase activity did not recover. Okadaic acid caused significant dose-dependent inhibition of adenylyl cyclase activity in intact PC-12 cells. Prolonged exposure of okadaic acid-treated PC-12 cells to adenosine agonists did not evoke further inhibition, suggesting that the inhibition of adenylyl cyclase activity during A2a desensitization may operate through a pathway that overlaps with the increased phosphorylation caused by okadaic acid. Inclusion of calcium in the adenylyl cyclase assay significantly inhibited cyclase activity. indicating that PC-12 cells contain Ca(2+)-inhibitable type VI adenylyl cyclase (AC6). This was confirmed by polymerase chain reaction-based detection of AC6 cDNA. Furthermore, incubation of PC-12 cell membrane fractions with purified protein phosphatase 2A or coexpression of protein phosphatase 2A with AC6 in COS-1 cells significantly increased AC6 activity. To reduce the possible influence of Gs alpha protein, we substituted guanosine-5'-O-(2-thio)diphosphate and MnCl2 for GTP and MgCl2, respectively, in some cyclase assays and found that the suppression of AC6 during A2a desensitization and okadaic acid treatment remained largely unchanged. Taken together, these data suggest that phosphorylation of AC6 might account for the inhibition of adenylyl cyclase activity during A2a desensitization in PC-12 cells.