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PubMed2004

Integration of ATP, cAMP, and Ca2+ signals in insulin granule exocytosis.

Intracellular ATP, cAMP, and Ca2+ are major signals involved in the regulation of insulin secretion in the pancreatic beta-cell. We recently found that the ATP-sensitive K+ channel (KATP channel) as an ATP sensor, cAMP-GEFII as a cAMP sensor, Piccolo as a Ca2+ sensor, and L-type voltage-dependent Ca2+ channel (VDCC) can interact with each other. In the present study, we examined the effects of cAMP and ATP on the interaction of cAMP-GEFII and sulfonylurea receptor-1 (SUR1). Interaction of cAMP-GEFII with SUR1 was inhibited by the cAMP analog 8-bromo-cAMP but not by ATP, and the inhibition by 8-bromo-cAMP persisted in the presence of ATP. In addition, SUR1, cAMP-GEFII, and Piccolo could form a complex. Piccolo also interacted with the alpha1 1.2 subunit of VDCC in a Ca2+-independent manner. These data suggest that the interactions of the KATP channel, cAMP-GEFII, Piccolo, and L-type VDCC are regulated by intracellular signals such as cAMP and Ca2+ and that the ATP, cAMP, and Ca2+ signals are integrated at a specialized region of pancreatic beta-cells.

Adenosine Triphosphate↗
PubMed1999

Increased intracellular cAMP renders HL-60 cells resistant to cytotoxicity of taxol.

The treatment of advanced cancers with paclitaxel (taxol) is hindered by the development of drug resistance. Resistance to taxol is known to be associated with multidrug resistance (MDR) and a mutation affecting either the alpha- or beta-subunit of tubulin. In this study, we demonstrated that an intracellular cAMP level may also play an important role in resistance to taxol in HL-60, acute promyelocytic leukemia cells. Exposure of HL-60 cells to various doses of taxol for 18 hr resulted in cell death. However, pretreatment of the cells with cAMP analogs such as N6:O2-dibutyl cAMP (Db-cAMP), 8-(4-chlorophenylthio) cAMP (CPT-cAMP) and 8-bromo-cAMP (8-Br-cAMP) or an intracellular cAMP elevating agent such as forskolin apparently rendered HL-60 cells more resistant to taxol, but not with dimethyl sulfoxide (DMSO) or retinoic acid (RA), well known differentiating agents. To investigate whether protein kinase A (PKA) activated by an increase in intracellular cAMP level could be involved in increased taxol resistance of the cells, we examined the effects of PKA inhibitors, including H-89 and KT5720, on taxol resistance induced by Db-cAMP. The PKA inhibitors significantly abolished Db-cAMP-induced taxol resistance. These results suggest that cAMP analogs may render tumor cells more resistant to taxol via PKA activation.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Camp for all connection: a community health information outreach project.

PURPOSE: The purpose of the Camp For All Connection project is to facilitate access to electronic health information resources at the Camp For All facility. SETTING/PARTICIPANTS/RESOURCES: Camp For All is a barrier-free camp working in partnership with organizations to enrich the lives of children and adults with chronic illnesses and disabilities and their families by providing camping and retreat experiences. The camp facility is located on 206 acres in Burton, Texas. The project partners are Texas Woman's University, Houston Academy of Medicine-Texas Medical Center Library, and Camp For All. BRIEF DESCRIPTION: The Camp For All Connection project placed Internet-connected workstations at the camp's health center in the main lodge and provided training in the use of electronic health information resources. A train-the-trainer approach was used to provide training to Camp For All staff. RESULTS/OUTCOME: Project workstations are being used by health care providers and camp staff for communication purposes and to make better informed health care decisions for Camp For All campers. EVALUATION METHOD: A post-training evaluation was administered at the end of the train-the-trainer session. In addition, a series of site visits and interviews was conducted with camp staff members involved in the project. The site visits and interviews allowed for ongoing dialog between project staff and project participants.

Chronic Disease↗
PubMed1975

Mechanisms of control for cAMP-dependent protein kinase from skeletal muscle.

A number of properties of homogeneous cyclic 3',5'-AMP (cAMP)-dependent protein kinase from rabbit skeletal muscle were determined. The enzyme is shown to be a tetramer consisting of one regulatory subunit dimer and two catalytic subunit monomers. Skeletal muscle protein kinase interacts with cAMP and MgATP as illustrated in the following equilibrium expression: R2C2 - (MgATP)2 + 2 cAMP in equilibrium R2 - (cAMP)2 + 2C + 2 MgATP. MgATP is shown to decrease the affinity of the enzyme for cAMP and to be necessary for recombination of the subunits. The concentration of the enzyme in tissue relative to that of cAMP is high enough to influence kinetic parameters of the activation process by cAMP. The cumulative effects of MgATP and high enzyme concentration are to increase the apparent activation constant for cAMP so that in vivo the enzyme would not be highly activated under basal conditions but would be greatly stimulated by increases in cAMP concentration. As a result, it is not necessary to invoke the concept of compartmentalization of cAMP to explain how it could regulate protein kinase activity in vivo. Finally, data are presented which indicate that a possible function of the heat-stable protein inhibitor of cAMP-dependent protein kinases may be to suppress the activity of protein kinase due to basal concentrations of cAMP. As such, the inhibitor could indirectly change the amount of cAMP needed to allow expression of protein kinase activity.

Adenosine Triphosphate↗
PubMed1991

Comparison of adenylate cyclase and cAMP-dependent protein kinase in gametocytogenic and nongametocytogenic clones of Plasmodium falciparum.

Adenylate cyclase and cAMP-dependent protein kinase activities in gametocytogenic (LE5) and nongametocytogenic (T9/96) clones of Plasmodium falciparum were compared to explore the role of cAMP in sexual differentiation of the parasite. Basal adenylate cyclase levels were equivalent in the 2 clones. However, cAMP-dependent histone II-A kinase activity was significantly higher in LE5 than in T9/96 over a range of cAMP concentrations. This difference was due to a decreased Vmax for the enzyme in the nongametocytogenic clone and not to an increased Ka for cAMP. Examination of parasite cAMP-binding proteins, likely to be kinase regulatory subunits, by both photoaffinity labeling with [32P]8-N3-cAMP and affinity chromatography of metabolically [35S]methionine-labeled cytosol of cAMP-agarose revealed a 53-kDa cAMP binding protein in both clones and a 49-kDa cAMP-binding protein in T9/96 that was absent in LE5. Our results suggest that T9/96 has lost the ability to undergo gametocytogenesis due to a substantial decrease in cAMP-dependent protein kinase activity rendering the parasite unable to respond to increased intracellular cAMP levels. Moreover, the reduction in cAMP-dependent protein kinase activity may be due to the presence of an alternative regulatory subunit of the kinase.

Adenylyl Cyclases↗
PubMed1993

Transplasma membrane redox system of HL-60 cells is controlled by cAMP.

Transplasma membrane redox activity of HL-60 cells was determined by measuring the prevention of ascorbate chemical oxidation. The ascorbate free radical produced as the first step of ascorbate oxidation was reduced back by the transplasma membrane electron transport system, causing then the regeneration of extracellular ascorbate. Agents that increase intracellular cAMP, such as forskolin and dibutyryl cAMP (db-cAMP), increased the rate of ascorbate regeneration by HL-60 cells. Also, the phosphodiesterase-resistant cAMP analogue Sp-cAMP-S (agonist of the protein kinase A) increased the electron flow to the ascorbate free radical at the plasma membrane. Rp-cAMP-S, antagonist of the protein kinase A, partially inhibited the redox activity of cells and abolished the effect of Sp-cAMP-S. Inhibition obtained after preincubation of cells in Rp-cAMP-S was reversed by Sp-cAMP-S. Tunicamycin, a compound that inhibited the electron flow to the ascorbate free radical at the plasma membrane, also reduced the response of transplasma membrane redox system to Sp-cAMP-S. Lactate slightly affected the ascorbate regeneration in nonstimulated cells, but showed a significant effect on Sp-cAMP-S-stimulated plasma membrane electron flow. We show here a role for cAMP in the short-term modulation of transplasma membrane redox system measured as the regeneration of ascorbate at the cell surface of HL-60 cells, probably mediated by cAMP-dependent protein kinases.

Ascorbic Acid↗
PubMed1996

Distribution and activation of cAMP- and cGMP-dependent protein kinases in highly purified human platelet plasma and intracellular membranes.

Previously cAMP- and cGMP-dependent protein kinases (cAMP-PK, cGMP-PK) have been found predominantly associated with the particulate fraction in human platelets. We now report the distribution and activation of cAMP-PK and cGMP-PK in highly purified fractions of human platelet plasma (PM) and intracellular membranes (IM) prepared using high voltage free flow electrophoresis. Two non-hydrolysable analogues of cAMP and cGMP namely Sp-5,6-DCI-cBiMPS and 8-p-CPT-cGMP have been used to activate cAMP-PK and cGMP-PK respectively. Addition of either agonist with [gamma 32P]ATP stimulated the endogenous activity of cAMP-PK or cGMP-PK in PM but not in IM. With PM Sp-5,6-DCI-cBiMPS stimulated the phosphorylation of protein substrates of Mr 16, 22, 24, 46-50, 66, 90, 160 and 250 kDa. A specific peptide inhibitor of cAMP-PK inhibited the phosphorylation of all of the substrates by Sp-5,6-DCI-cBiMPS. 8-pCPT-cGMP also induced the phosphorylation of a number of substrates particularly 16, 22, 46-50, 90 and 250 kDa proteins. Inclusion of the cAMP-PK inhibitor peptide totally blocked the phosphorylation of the 16 and 22 kDa proteins, partially inhibited phosphorylation of 46-50 and 90 kDa proteins and had no effect on the 250 kDa protein indicating the 46-50, 90 and 250 kDa proteins were also cGMP-PK substrates. Western blotting with antibodies to cGMP-PK and the catalytic subunit of cAMP-PK revealed the presence of the kinases to be exclusively associated with PM with no detection in IM. The presence of cAMP-PK substrates in IM was investigated by exogenous addition of catalytic subunit of cAMP-PK. Phosphoproteins of Mr 16, 22, 27, 30, 45, 75, 116 and 250 kDa were detected. A range of antibodies to cAMP-PK substrates were used to identify and localise the substrates. These antibodies revealed GPIb and VASP to be exclusively associated with PM fractions. Rap IB was also predominantly associated with PM with a small level detected in IM. Antibodies to the IP3 receptor (18A 10 and 4C11) revealed the protein to be predominantly associated with IM. Additionally the antibody 4C11 recognised a 230 kDa protein band in PM that was not seen in IM. From the known specificity of these antibodies the results confirm the presence of a type 1 IP3 receptor in IM and a distinct (possible type III) IP3 receptor with the PM. The 16, 22, 27, 30, 75 and 116 kDa proteins in IM represent newly detected substrates for cAMP-PK of presently unknown identity.

Blood Platelets↗
PubMed2006

Improved perceived health status persists three months after a special sports camp for children with congenital heart disease.

INTRODUCTION: Although summer and sports camps for children with congenital heart disease are organized in many countries and regions, empirical data on the effects of such camps is limited. OBJECTIVES: The aim of the present study was to investigate changes in the perceived health status and habitual physical activities in children attending a special sports camp. MATERIALS AND METHODS: In this longitudinal study, 25 children with congenital heart disease who participated in a three-day multi-sports camp were included. The perceived health status was measured using the Child Health Questionnaire-Child Form, CHQ-CF87, completed by the child at the start of the camp (T1), at the end of the camp (T2), and 3 months after the camp concluded (T3). Habitual physical activities were assessed by means of a modified version of the Baecke questionnaire, which was completed by one of the parents at T1 and T3. RESULTS: During the sports camp, we observed significant improvements in the children's perception of their physical functioning, role-physical functioning, general health, role-emotional functioning, self-esteem, mental health, and general behavior. For physical functioning, role-emotional functioning, and family activities, high scores persisted three months after the sports camp concluded. The habitual physical activities (sport and leisure time) of the children remained unchanged. DISCUSSION AND CONCLUSION: In conclusion, we propose that a special sports camp for children with congenital heart disease may improve specific dimensions of subjective health status. Our study confirms a previous report on the benefits of such camps for afflicted children. If these findings can be further corroborated in other settings, participation in sport camps should be advocated as a simple, noninvasive means to promote healthier children.

Adolescent↗
PubMed2005

Metabolic control and educational status in children with type 1 diabetes: effects of a summer camp and intensive insulin treatment.

Our aim was to evaluate prospectively, in our diabetic patients, the impacts of a summer camp and intensive insulin treatment (IIT) on both metabolic control and disease-related educational level. Twenty-five patients participated in a 7-day-long summer camp. Before the camp, all patients were on therapy with short-acting human insulin (SAI) and intermediate-acting insulin (IAI) twice daily. On arrival, their insulin therapy regimen was changed by IIT including either SAI or rapid-acting insulin analogue (RAI) three times before meals supplemented by IAI at bedtime. Following the camp, all participants were given IIT with RAI plus IAI. Frequency of hypoglycaemia, insulin dose, body mass index (BMI) and glycohaemoglobin (HbA1c) levels were assessed at pre-camp and post-camp controls. To evaluate the effectiveness of camp-assisted education, all participants were regularly tested. We observed significant elevations in total daily dose of insulin and BMI at months 3 and 6 when compared with the pre-camp values but, by month 12, they were not significantly different from precamp values. The mean HbA(1c) level decreased significantly at months 6 and 12. Severe hypoglycaemic episodes and ketoacidosis were not detected during the camp and the following year. Significant improvements in knowledge about diabetes and self-management were determined at the end of the camp, after 6 and 12 months. Camp-assisted IIT with RAI improved metabolic control of diabetic children. Additionally, camp-assisted education has a positive effect on disease-related educational level and self-management.

Adolescent↗
PubMed2000

Summer camps for diabetic children: an experience in Antalya, Turkey.

To evaluate the effectiveness of diabetic summer camps with objective parameters, we examined the data relative to summer camps organized by our department in Antalya in the last two years. The duration of the camps was 10 days. Twenty-eight diabetic children with an average age of 13.6 +/- 2.9 years (range 8-20) participated in the first camp, fourteen of whom participated in both camps. The medical personnel consisted of three pediatric endocrinologists, one psychologist, two diabetes nurses and two dietitians. Despite a mean 10% reduction in insulin dosage and 10% increment in daily calorie intake at the beginning of the camp, hypoglycemia was common (mean, 2.4 hypoglycemic episodes per subject). Ketoacidosis was not encountered in any of the subjects during and after camps. An increment in weight in children whose weights, with respect to heights, were under the ideal weight and a decrement in weight of overweight children were observed at the end of the first camp. A significant improvement in knowledge and self-management of the disease was noted at the end of the camps. Improvement in nutrition and diabetic knowledge level of the children who participated in these consecutive camps was more obvious in the second compared with that in the first camp. No significant change in HbA1c level was observed at follow-up. In conclusion, summer camps are an invaluable way for diabetic children to gain skills in managing their disease.

Adolescent↗
PubMed1998

Involvement of cAMP-dependent protein kinase in the nucleus accumbens in cocaine self-administration and relapse of cocaine-seeking behavior.

cAMP-dependent protein kinase (PKA) in the nucleus accumbens (NAc) has been implicated in cocaine addiction because (1) cocaine reinforcement is mediated by dopamine receptors that modulate cAMP formation, and (2) repeated exposure to cocaine upregulates the cAMP system in NAc neurons. This study tested PKA involvement in cocaine self-administration and relapse of cocaine-seeking behavior by infusing cAMP analogs that activate or inhibit PKA into the NAc of rats. Bilateral intra-NAc infusions of the PKA inhibitor Rp-cAMPS reduced baseline cocaine self-administration, shifted the dose-response curve for cocaine self-administration to the left, and induced relapse of cocaine-seeking behavior after extinction from cocaine self-administration, consistent with an enhancement of cocaine effects in each paradigm. In contrast, pretreatment with intra-NAc infusions of a PKA activator, Sp-cAMPS or dibutyryl cAMP, increased baseline cocaine self-administration during the second hour of testing and shifted the dose-response curve to the right, consistent with an antagonist-like action. After extinction from cocaine self-administration, similar infusions of Sp-cAMPS induced generalized responding at both drug-paired and inactive levers. As an index of PKA activity in vivo, NAc infusions of Rp-cAMPS reduced basal levels of dopamine-regulated phosphoprotein-32 phosphorylation and blocked amphetamine-induced increases in cAMP response element-binding protein (CREB) phosphorylation. Conversely, NAc infusions of Sp-cAMPS increased phosphorylation of CREB. Together, these results suggest that sustained upregulation of the cAMP system in the NAc after repeated cocaine exposure could underlie tolerance to cocaine reinforcement, whereas acute inhibition of this system may contribute to drug craving and relapse in addicted subjects.

Animals↗
PubMed1986

Increased level of cAMP-dependent protein kinase in aging human lung fibroblasts.

Regulation of the expression of cAMP-dependent protein kinase in cellular aging was studied using the IMR-90 diploid human lung fibroblasts. The level of cAMP-dependent protein kinase present in cell extracts was monitored by 1) photoactivated incorporation of 8-N3-[32P]cAMP into the 47,000- and 54,000-dalton regulatory subunits of the type I and type II cAMP-dependent protein kinases, respectively; 2) cAMP-dependent phosphorylation of histone II AS catalyzed by the catalytic subunit of the kinase; and 3) fractionation and analysis of the type I and type II cAMP-dependent protein kinase by DEAE-Sephacel column chromatography. Our results showed an approximately two- to threefold increase in the level of the type I cAMP-dependent protein kinase and a somewhat smaller increase in the type II kinase in extracts of the "old" IMR-90 cells (population doubling greater than 48) as compared to that of the "young" cells (PDL 22-27). The timing of the increase in cAMP-dependent protein kinase coincided with a significant decrease in the proliferative potential of the cells. This result together with previously demonstrated effects of cAMP in the control of cell growth and differentiation and the increased expression of cAMP-dependent protein kinase during terminal differentiation of the murine preadipocytes (3T3-L1) and myoblast (L-5, L-6, and C2C13) suggests that regulation of the levels of cAMP and cAMP-dependent protein kinase plays a significant role in the control of cell growth and differentiation.

Cell Division↗
PubMed1991

Modulation of responsiveness to cAMP stimulating agonists by phorbol ester in fetal rat osteoblasts.

We studied the effect of activation of protein kinase C (PKC) by a phorbol ester on cAMP accumulation in fetal rat osteoblasts. Activation of PKC by phorbol 12-myristate 13-acetate (PMA) caused a potentiation of cAMP accumulation induced by parathyroid hormone (PTH), forskolin, and cholera toxin. The results suggest that the potentiating effect of PMA on PTH-induced cAMP accumulation was not due to an effect on the PTH-receptor nor to an effect on cAMP degradation, as the effect of PMA persisted in the presence of a phosphodiesterase inhibitor. Pretreatment of the cells with pertussis toxin did not prevent the action of PMA, indicating that PMA does not act via the inhibitory G-protein. PMA had a biphasic effect on prostaglandin E2 (PGE2)-induced cAMP accumulation; i.e., at concentrations greater than or equal to 10(-6) M, PMA potentiated the PGE2-induced cAMP response but PMA attenuated cAMP accumulation induced by concentrations of PGE2 less than or equal to 5.10(77) M. From our data we conclude that PKC can interact with a stimulated cAMP pathway in a stimulatory and inhibitory manner. Potentiation of cAMP accumulation is probably due to modification of the adenylate cyclase complex, whereas attenuation of stimulated cAMP accumulation appears to be due to an effect on a different site of the cAMP generating pathway, which may be specific to PGE2-induced cAMP accumulation.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Contribution of the carboxyl-terminal regional of the cAMP-dependent protein kinase type I alpha regulatory subunit to cyclic nucleotide interactions.

The carboxyl-terminal 19 amino acids of the type I alpha regulatory subunit (RI alpha) of cAMP-dependent protein kinase (PKA) were investigated to determine their contributions to cAMP selectivity. The parent RI alpha subunit contained an Ala to Thr mutation at position 334 so that it would bind both cAMP and cGMP with high affinity. Stop codons were introduced into the parent cDNA construct at positions corresponding to Val-375, Asn-372, Gln-370, and Cys-360. The purified, bacterially expressed proteins were characterized for their cAMP and cGMP dissociation properties. Site-selective cAMP analogs were used to compete against [3H]cAMP binding to the mutant RI alpha subunits to correctly assign fast and slow dissociation t1/2 values to the A and B domains. A greater than 60-fold drop in B domain t1/2 in the Asn-372-stop to Gln-370-stop transition implicated Tyr-371 as an important cAMP-binding determinant. A similar drop in [3H]cGMP t1/2 for the same transition suggested that the cGMP/cAMP selectivity was not altered. To test this further, Tyr-371 was mutated to Ala, Phe, and Arg in the parent construct. The cAMP and cGMP t1/2 values were determined, as were protein kinase activation constants (Ka) for holoenzymes formed from mutant RI alpha subunits and purified catalytic subunit. The Ka data suggested that mutation of Tyr-371 enhanced B domain cAMP selectivity. Isolated B domains containing Tyr-371-Arg or Tyr-371-Phe mutations were constructed, expressed, and purified to determine their relative inhibition constants (K'I) for cGMP vs cAMP. These data showed that B domain cAMP selectivity was minimally affected by alteration of Tyr-371. Based on these results, it is concluded that aromatic stacking is not important for determining B-domain cyclic nucleotide selectivity. It is proposed that the main function of Tyr-371 is stabilization of the B-domain cAMP-binding pocket through hydrogen bonding with Glu-324.

Amino Acid Sequence↗
PubMed1994

Costimulatory signals modulate the antiproliferative effects of agents that elevate cAMP in T cells.

Stimulation of highly purified human T cells with immobilized anti-CD3 monoclonal antibody (mAb) in the presence of cAMP-inducing agents results in inhibition of proliferation by these T cells. In the present study, experiments were performed to determine how costimulatory signals modulate the inhibitory effects of cAMP-elevating agents on proliferation and interleukin 2 (IL-2) secretion by anti-CD3 mAb-stimulated T cells. Accordingly, the level of anti-CD3 mAb-induced T cell proliferation was determined in the presence or absence of accessory cells, anti-CD28 mAb, or phorbol myristic acid (PMA) in the presence of the adenylyl cyclase (AC)-linked receptor agonists prostaglandin E2 (PGE2), or isoproterenol (ISO) as well as the AC activator forskolin (FSK) or the cAMP analog dibutyryl-cAMP (dB-cAMP). While all three costimulators enhanced the level of anti-CD3 mAb-induced T cell proliferation and IL-2 secretion, they were variable in their ability to overcome the immunosuppressive effects of the cAMP elevating agents. The order of potency of the costimulatory signals in reversing the inhibitory effects of cAMP-elevating agents on anti-CD3 mAb-induced T cell proliferation and IL-2 secretion was PMA > accessory cells > anti-CD28 mAb. Differences were noted in the ability of the costimulatory signals to overcome the immunosuppressive effects of the various cAMP-inducing agents. Thus, the effects of PGE2 or ISO on T cell proliferation or IL-2 secretion were more readily overcome by costimulatory signals than those elicited by FSK or dB-cAMP. Experiments designed to investigate the mechanisms involved in these effects showed that neither accessory cells nor anti-CD28 mAb altered the level of cAMP accumulation or protein kinase A (PKA) activity in T cells stimulated with cAMP-elevating agents. However, PMA was found to decrease both cAMP accumulation and PKA activity in T cells stimulated with PGE2 or ISO but not FSK. These results suggest that the overall immunosuppressive effects of naturally occurring substances such as PGE2 or catecholamines may be altered by costimulatory signals when antigen-specific T cells interact with antigen-presenting cells.

Adenylyl Cyclases↗
PubMed1994

Expression of hepatocyte growth factor is up-regulated through activation of a cAMP-mediated pathway.

Hepatocyte growth factor (HGF) is a multifunctional cytokine with mitogenic, motogenic, morphogenic, and tumor-suppressing activities. Despite the broad spectrum of its biological activities, HGF is most likely the physiological hepatotrophic factor that triggers or modulates liver regeneration. Regulatory mechanisms for HGF production are crucial for understanding the control of liver regeneration. We previously reported that HGF production by human skin fibroblasts is stimulated by a protein kinase C (PKC)-mediated pathway. We determined here whether gene expression and production of HGF in human skin fibroblasts can be induced via activation of a cAMP-mediated pathway. HGF secretion by the cells was markedly stimulated by the cAMP-elevating agents, forskolin, cholera toxin, prostaglandin E2 (PGE2), and 3-isobutyl-1-methylxanthine, as well as by the membrane-permeable cAMP analogues, 8-bromo-cAMP and dibutyryl cAMP. The dose-response curves of induction of HGF secretion by cholera toxin and forskolin were nearly parallel with those of the intracellular cAMP levels. HGF mRNA levels did not significantly increase at 5 and 10 h, but increased considerably 15 h or more after the addition of cholera toxin. Forskolin, 8-bromo-cAMP, and PGE2 also caused appreciable up-regulation of HGF gene expression with a similar time course. Although human skin fibroblasts of various origins secreted variable amounts of HGF, the cAMP-elevating agents and the cAMP analogues caused a very marked increase in HGF production in all of them. The agents also enhanced highly active HGF secretion by MRC-5 human embryonic lung fibroblasts. Dexamethasone and transforming growth factor-beta 1, which inhibit PKC-mediated HGF secretion, down-regulated HGF mRNA expression and HGF production in the cells treated with the cAMP-elevating agents and the cAMP analogues. These results indicate that HGF expression in human skin fibroblasts is stimulated by activation of a cAMP-mediated pathway.

1-Methyl-3-isobutylxanthine↗
PubMed1995

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells.

G-protein coupled Angiotensin II receptors (AT1A), mediate cellular responses through multiple signal transduction pathways. In AT1A receptor-transfected CHO-K1 cells (T3CHO/AT1A), angiotensin II (AII) stimulated a dose-dependent EC50 = 3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT1, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca2+ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or ca2+/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [3H]thymidine incorporation in T3CHOA/AT1A cells. AII (1 microM) significantly inhibited cell number (51% at 96 h) and [3H]thymidine incorporation of 68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT1 receptor. Forskolin (10 microM) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [3H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity an tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT1A cells, in particular at 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.

Angiotensin II↗
PubMed1988

Vasoactive hormones and cAMP affect pericyte contraction and stress fibres in vitro.

Pericytes are contractile cells of the microvascular wall that may influence capillary haemodynamics and permeability. We examined the contractile responses of cultured pericytes to selected vasoactive agents and cAMP agonists. Morphological and biochemical changes associated with these responses were also studied. Pericytes seeded onto silicone rubber contracted when stimulated with histamine or serotonin, relaxed in response to the beta-adrenergic agonist isoproterenol and did not respond to epinephrine. Since hormonal-induced relaxation of vascular smooth muscle involves cAMP, we investigated the ability of cAMP, to modulate pericyte contraction. Dibutyryl cAMP and forskolin (an adenylate cyclase activator) both induced pericyte relaxation and elevated intracellular cAMP levels. Isoproterenol increased cAMP levels but epinephrine had no effect. However, when epinephrine and isoproterenol were co-incubated with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), cAMP was increased to levels above those elicited by these agonists alone. Serotonin and histamine in the presence of IBMX did not affect cAMP levels. These results suggest that certain vasoactive agents may relax pericytes by cAMP-dependent processes. We have shown previously that stress fibres are also involved in pericyte contraction. Hence, changes in the staining patterns of stress fibres in response to these selected agonists were studied. Histamine, serotonin and epinephrine had no apparent effect on stress fibre staining. Dibutyryl cAMP, forskolin, and isoproterenol, which relax pericytes and increase cAMP, disassembled fibres. In summary, the results demonstrate that the contractile activity of cultured pericytes in vitro can be regulated by vasoactive agonists and that changes in cAMP and stress fibres may mediate the regulation.

Actin Cytoskeleton↗