Cyclic adenosine phosphate improves functional recovery after spinal cord injury via activating unfolded protein response.

The current study aimed to evaluate the role and underlying mechanism of cyclic adenosine phosphate (cAMP) on the functional recovery of spinal cord injury (SCI). Basso, Beattie and Bresnahan (BBB) scoring and inclined plane test indicated that cAMP treatment improved the functional recovery of SCI rats. Real time PCR and western blot analysis showed the mRNA and protein levels of IRE1, PERK, and ATF6 were increased in the SCI rats than those of sham control. However, higher levels of IRE1, PERK, and ATF6 were indicated after cAMP treatment. Meanwhile, more apoptotic cells were observed in the SCI rats, as evidenced by TUNEL staining and increased expression of GRP78, CHOP, and caspase12. In contrast, the expression of GRP78, CHOP, and caspase12 was decreased in SCI rats after cAMP treatment. In summary, we showed novel data that cAMP reduced cell apoptosis and functional recover after SCI mainly via activating UPR.

A novel adenosine precursor 2',3'-cyclic adenosine monophosphate inhibits formation of post-surgical adhesions.

BACKGROUND: Intraperitoneal adenosine reduces abdominal adhesions. However, because of the ultra-short half-life and low solubility of adenosine, optimal efficacy requires multiple dosing. AIM: Here, we compared the ability of potential adenosine prodrugs to inhibit post-surgical abdominal adhesions after a single intraperitoneal dose. METHODS: Abdominal adhesions were induced in mice using an electric toothbrush to damage the cecum. Also, 20 µL of 95 % ethanol was applied to the cecum to cause chemically induced injury. After injury, mice received intraperitoneally either saline (n = 18) or near-solubility limit of adenosine (23 mmol/L; n = 12); 5'-adenosine monophosphate (75 mmol/L; n = 11); 3'-adenosine monophosphate (75 mmol/L; n = 12); 2'-adenosine monophosphate (75 mmol/L; n = 12); 3',5'-cyclic adenosine monophosphate (75 mmol/L; n = 19); or 2',3'-cyclic adenosine monophosphate (75 mmol/L; n = 20). After 2 weeks, adhesion formation was scored by an observer blinded to the treatments. In a second study, intraperitoneal adenosine levels were measured using tandem mass spectrometry for 3 h after instillation of 2',3'-cyclic adenosine monophosphate (75 mmol/L) into the abdomen. RESULTS: The order of efficacy for attenuating adhesion formation was: 2',3'-cyclic adenosine monophosphate > 3',5'-cyclic adenosine monophosphate ≈ adenosine > 5'-adenosine monophosphate ≈ 3'-adenosine monophosphate ≈ 2'-adenosine monophosphate. The groups were compared using a one-factor analysis of variance, and the overall p value for differences between groups was p < 0.000001. Intraperitoneal administration of 2',3'-cAMP yielded pharmacologically relevant levels of adenosine in the abdominal cavity for >3 h. CONCLUSION: Administration of 2',3'-cyclic adenosine monophosphate into the surgical field is a unique, convenient and effective method of preventing post-surgical adhesions by acting as an adenosine prodrug.

Endogenous production of extracellular adenosine by trabecular meshwork cells: potential role in outflow regulation.

PURPOSE: To investigate the mechanisms for endogenous production of extracellular adenosine in trabecular meshwork (TM) cells and evaluate its physiological relevance to the regulation of aqueous humor outflow facility. METHODS: Extra-cellular levels of adenosine monophosphate (AMP) and adenosine in porcine trabecular meshwork (PTM) cells treated with adenosine triphosphate (ATP), AMP, cAMP or forskolin with or without specific inhibitors of phosphodiesterases (IBMX) and CD73 (AMPCP) were determined by high-pressure liquid chromatography fluorometry. Extracellular adenosine was also evaluated in cell cultures subjected to cyclic mechanical stress (CMS) (20% stretching; 1 Hz) and after disruption of lipid rafts with methyl-ß-cyclodextrin. Expression of CD39 and CD73 in porcine TM cells and tissue were examined by Q-PCR and Western blot. The effect of inhibition of CD73 on outflow facility was evaluated in perfused living mouse eyes. RESULTS: PTM cells generated extracellular adenosine from extracellular ATP and AMP but not from extracellular cAMP. Increased intracellular cAMP mediated by forskolin led to a significant increase in extracellular adenosine production that was not prevented by IBMX. Inhibition of CD73 resulted, in all cases, in a significant decrease in extracellular adenosine. CMS induced a significant activation of extracellular adenosine production. Inhibition of CD73 activity with AMPCP in living mouse eyes resulted in a significant decrease in outflow facility. CONCLUSIONS: These results support the concept that the extracellular adenosine pathway might play an important role in the homeostatic regulation of outflow resistance in the TM, and suggest a novel mechanism by which pathologic alteration of the TM, such as increased tissue rigidity, could lead to abnormal elevation of IOP in glaucoma.

Development and applications of a broad-coverage, TR-FRET-based kinase binding assay platform.

The expansion of kinase assay technologies over the past decade has mirrored the growing interest in kinases as drug targets. As a result, there is no shortage of convenient, fluorescence-based methods available to assay targets that span the kinome. The authors recently reported on the development of a non-activity-based assay to characterize kinase inhibitors that depended on displacement of an Alexa Fluor 647 conjugate of staurosporine (a "tracer") from a particular kinase. Kinase inhibitors were characterized by a change in fluorescence lifetime of the tracer when it was bound to a kinase relative to when it was displaced by an inhibitor. Here, the authors report on improvements to this strategy by reconfiguring the assay in a time-resolved fluorescence resonance energy transfer (TR-FRET) format that simplifies instrumentation requirements and allows for the use of a substantially lower concentration of kinase than was required in the fluorescence-lifetime-based format. The authors use this new assay to demonstrate several aspects of the binding assay format that are advantageous relative to traditional activity-based assays. The TR-FRET binding format facilitates the assay of compounds against low-activity kinases, allows for the characterization of type II kinase inhibitors either using nonactivated kinases or by monitoring compound potency over time, and ensures that the signal being detected is specific to the kinase of interest and not a contaminating kinase.

Optical molecular imaging of epidermal growth factor receptor expression to improve detection of oral neoplasia.

BACKGROUND: The development of noninvasive molecular imaging approaches has the potential to improve management of cancer. METHODS: In this study, we demonstrate the potential of noninvasive topical delivery of an epidermal growth factor-Alexa 647 (EGF-Alexa 647) conjugate to image changes in epidermal growth factor receptor expression associated with oral neoplasia. We report a series of preclinical analyses to evaluate the optical contrast achieved after topical delivery of EGF-Alexa 647 in a variety of model systems, including cells, three-dimensional tissue cultures, and intact human tissue specimens using wide-field and high-resolution fluorescence imaging. Data were collected from 17 different oral cancer patients: eight pairs of normal and abnormal biopsies and nine resected tumors were examined. RESULTS: The EGF-dye conjugate can be uniformly delivered throughout the oral epithelium with a penetration depth exceeding 500 microm and incubation time of less than 30 minutes. After EGF-Alexa 647 incubation, the presence of oral neoplasia is associated with a 1.5- to 6.9-fold increase in fluorescence contrast compared with grossly normal mucosa from the same patient with both wide-field and high-resolution fluorescence imaging. CONCLUSIONS: Results illustrate the potential of EGF-targeted fluorescent agents for in vivo molecular imaging, a technique that may aid in the diagnosis and characterization of oral neoplasia and allow real-time detection of tumor margins.

Distribution, amplification, and summation of cyclic nucleotide sensitivities within single olfactory sensory cilia.

Submicron local cAMP elevation was used to map the distribution of transduction channels in single olfactory cilia. After the fine fluorescent visualization of the cilium with the laser-scanning confocal microscope, the intraciliary cAMP was jumped locally with the laser beam that photolyzes cytoplasmic caged compounds. Simultaneously, cells' responses were obtained with the whole-cell patch clamp. Responses were observed anywhere within the cilia, showing the broad distribution of transduction channels. For odor detection, such distribution would be useful for expanding the available responding area to increase the quantum efficiency. Also, the stimulus onto only 1 microm region induced >100 pA response operated by >700-2300 channels, although only 1 pA is sufficient for olfactory cells to generate action potentials. The large local response indicates a presence of strong amplification achieved with a high-density distribution of the transduction channels for the local ciliary excitation.

A unique function for LRP-1: a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes.

BACKGROUND: Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated, clathrin-dependent mechanism to form the unique platelet-derived FV pool. OBJECTIVE: The role of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), or a related family member, in FV endocytosis by megakaryocytes was examined because of its known interactions with other proteins involved in hemostasis. METHODS: LRP-1 expression by megakaryocytes and its functional role in FV endocytosis was confirmed using reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies. FV binding to megakaryocytes was performed under Ca(2+)-free conditions to quantify binding in the absence of endocytosis. RESULTS AND CONCLUSION: Cell surface expression of LRP-1 by CD34+ ex vivo-derived megakaryocytes and the megakaryocyte-like cell line CMK was confirmed using anti-LRP-1 antibodies and was consistent with the detection of LRP-1 message in these cells. All cells capable of endocytosing FV expressed LRP-1. Anti-LRP-1 antibodies and receptor-associated protein (RAP), a known antagonist of LDL receptor family members, displaced only 50% of the [(125)I]FV bound to megakaryocytes. FV binding to megakaryocytes showed positive cooperativity (Hill coefficient = 1.92 +/- 0.18) that was substantially reduced in the presence of RAP (1.47 +/- 0.26). As FV endocytosis is specific to this cofactor, a model is hypothesized where FV binding to a specific receptor facilitates binding and endocytosis of a second FV molecule by LRP-1, or a related family member. These combined observations describe a unique role for LRP-1 in endocytosis of a coagulation protein trafficked to alpha-granules and not destined for lysosomal degradation.

cAMP-adenosine pathway in the proximal tubule.

The "extracellular cAMP-adenosine pathway" refers to the conversion of cAMP to AMP by ecto-phosphodiesterase, followed by metabolism of AMP to adenosine by ecto-5'-nucleotidase, with all the steps occurring in the extracellular compartment. This study investigated whether the extracellular cAMP-adenosine pathway exists in proximal tubules. Freshly isolated proximal tubules rapidly converted basolaterally administered cAMP to AMP and adenosine. Proximal tubular cells in culture (first passage) rapidly converted apically administered cAMP to AMP and adenosine. In both freshly isolated proximal tubules and cultured proximal tubular cells, conversion of cAMP to AMP and adenosine was affected by a broad-spectrum phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine), an ecto-phosphodiesterase inhibitor (1,3-dipropyl-8-p-sulfophenylxanthine), and a blocker of ecto-5'-nucleotidase (alpha,beta-methyleneadenosine-5'-diphosphate) in a manner consistent with exogenous cAMP being processed by the extracellular cAMP-adenosine pathway. In cultured proximal tubular cells, but not freshly isolated proximal tubules, stimulation of adenylyl cyclase increased extracellular concentrations of cAMP, AMP, and adenosine plus inosine, and these changes were also modulated by the inhibitors in a manner consistent with the extracellular cAMP-adenosine pathway. Conversion of renal interstitial (basolateral) cAMP and AMP to adenosine in vivo was shown by microdialysis coupled with ion trap mass spectrometry. Western blot analysis showed A1, A2A, and A3 receptors on both apical and basolateral proximal tubular membranes, with A1 and A2A receptors more highly expressed on basolateral compared with apical membranes. We conclude that cAMP that reaches either the apical or basolateral membranes of proximal tubular cells is converted in part to adenosine that has ready access to adenosine receptors.

Uptake of 3H-cAMP by retinal pigment epithelium isolated from bluegill sunfish (Lepomis macrochirus).

BACKGROUND: In bluegill sunfish, the melanin-containing pigment granules of the retinal pigment epithelium undergo cyclic movements in response both to ambient lighting and circadian cues. Pigment granules aggregate into the cell body at night (in the dark), and disperse into apical processes during the day (in the light). Regulation of pigment granule aggregation in a number of fishes depends on modulating the intracellular levels of cyclic adenosine monophosphate. RESULTS: Here we show isolated RPE takes up cyclic adenosine monophosphate (cAMP) in a saturable manner, exogenously applied cAMP induces pigment granule aggregation in retinal pigment epithelium isolated from bluegill, and aggregation induced in this manner is inhibited by treatment with probenecid, an organic anion transport inhibitor. CONCLUSION: Our results raise the possibility that cAMP functions as a messenger secreted from the neural retina to signal darkness to the RPE, which takes it up. It further suggests that organic anion transport systems are the route by which cAMP crosses RPE cell membranes since probenecid inhibits extracellular cAMP from causing pigment granule aggregation.

D2 dopamine receptor-induced sensitization of adenylyl cyclase type 1 is G alpha(s) independent.

Acute activation of D2 dopamine receptors inhibits adenylyl cyclase (EC 4.6.1.1), whereas persistent activation of these inhibitory receptors results in a compensatory increase in cyclic AMP accumulation. This sensitization of adenylyl cyclase is thought to involve enhanced Galpha(s)-adenylyl cyclase interactions; however, the absolute requirement of Galpha(s) has not been determined. The present study used a Galpha(s)-deficient cell line to examine directly the role of Galpha(s) in D2 dopamine receptor-induced sensitization of recombinant adenylyl cyclase type 1 (AC1) and 5 (AC5). In acute experiments, quinpirole activation of the D2 dopamine receptor inhibited AC1 and AC5 activity, indicating that the acute regulatory properties of AC1 and AC5 were retained in the absence of Galpha(s). Subsequent experiments revealed that short-term (2 h) activation of the D2 dopamine receptor resulted in significantly enhanced forskolin-stimulated AC1 activity in the absence of Galpha(s), whereas sensitization of forskolin-stimulated AC5 activity appeared to require Galpha(s). The Galpha(s)-independent sensitization of AC1 was explored further using AC1-selective activation protocols (A23187 and CCE) following short- and long-term agonist treatment. These studies revealed that persistent activation of D2 dopamine receptors sensitized AC1 activity to Ca2+ stimulation in cells devoid of endogenous Galpha(s) and demonstrate directly that sensitization of AC1 is Galpha(s)-independent.

Extracellular cyclic AMP-adenosine pathway in isolated adipocytes and adipose tissue.

OBJECTIVE: Our goal was to evaluate the presence and lipolytic impact of the extracellular cyclic adenosine monophosphate (AMP)-adenosine pathway in adipose tissue. RESEARCH METHODS AND PROCEDURES: Sixteen miniature Yucatan swine (Sus scrofa) were used for these in vitro and in situ experiments. Four microdialysis probes were implanted into subcutaneous adipose tissue and perfused at 2 microL/min with Ringer's solution containing no addition, varying levels of cyclic AMP, 10 microM isoproterenol, or 10 microM isoproterenol plus 1 mM alpha,beta-methylene adenosine 5'-diphosphate (AMPCP), a 5'-nucleotidase inhibitor. Dialysate was assayed for AMP, adenosine, inosine, hypoxanthine, and glycerol. Freshly isolated adipocytes were incubated with buffer, 1 microM isoproterenol, or 1 microM isoproterenol plus 0.1 mM AMPCP, and extracellular levels of AMP, adenosine, inosine, hypoxanthine, and glycerol were measured. RESULTS: Perfusion of adipose tissue with exogenous cyclic AMP caused a significant increase in AMP and adenosine appearance. Perfusion with AMPCP, in the presence or absence of isoproterenol, significantly increased the levels of AMP and glycerol, whereas it significantly reduced the level of adenosine and its metabolites. However, the AMPCP-provoked increase in lipolysis observed in situ and in vitro was not temporally associated with a decrease in adenosine. DISCUSSION: These data suggest the existence of a cyclic AMP-adenosine pathway in adipocytes and adipose tissue. The role of this pathway in the regulation of lipolysis remains to be clarified.

Brain region specific alterations in the protein and mRNA levels of protein kinase A subunits in the post-mortem brain of teenage suicide victims.

Protein kinase A (PKA), a critical component of the adenylyl cyclase signaling system, phosphorylates crucial proteins and has been implicated in the pathophysiology of depression and suicide. The objective of the study was to examine if changes in PKA activity or in the protein and messenger RNA (mRNA) expression of any of its subunits are related to the pathophysiology of teenage suicide. We determined PKA activity and the protein and mRNA expression of different subunits of PKA in cytosol and membrane fractions obtained from the prefrontal cortex, (PFC) hippocampus, and nucleus accumbens (NA) of post-mortem brain from 17 teenage suicide victims and 17 nonpsychiatric control subjects. PKA activity was significantly decreased in the PFC but not the hippocampus of teenage suicide victims as compared with controls. However, the protein and mRNA expression of only two PKA subunits, that is, PKA RIalpha and PKA RIbeta, but not any other subunits were significantly decreased in both membrane and cytosol fractions of the PFC and protein expression of RIalpha and RIbeta in the NA of teenage suicide victims as compared to controls. A decrease in protein and mRNA expression of two specific PKA subunits may be associated with the pathogenesis of teenage suicide, and this decrease may be brain region specific, which may be related to the specific behavioral functions associated with these brain areas. Whether these changes in PKA subunits are related to suicidal behavior or are a result of suicide or are specific to suicide is not clear at this point.

Uptake of liposomally entrapped adenosine-3'-5'-cyclic monophosphate in mouse brain.

[3H] Adenosine-3',5'-cyclic monophosphate (cAMP) could be entrapped efficiently into small unilamellar vesicles when bound to cAMP-dependent protein kinase. The leakage of [3H]cAMP protein kinase complex from liposomes was reduced by more than 60% as compared to free [3H]cAMP. Hyperosmolar mannitol increased the delivery of liposomally entrapped [3H]cAMP protein kinase to the brain with maximum uptake occurring at 10 min after mannitol administration. Optimal delivery to the brain was observed when vesicles composed of total brain lipids or phosphatidylcholine:cholesterol:sulfatides (7:2:1) were used. A slower clearance of liposomally entrapped material from brain tissue was seen under hyperosmolar conditions.

Cyclic GMP protein kinase activity is reduced in thyroxine-induced hypertrophic cardiac myocytes.

1. We tested the hypothesis that the cGMP-dependent protein kinase has major negative functional effects in cardiac myocytes and that the importance of this pathway is reduced in thyroxine (T4; 0.5 mg/kg per day for 16 days) hypertrophic myocytes. 2. Using isolated ventricular myocytes from control (n = 7) and T4-treated (n = 9) rabbit hypertrophic hearts, myocyte shortening was studied with a video edge detector. Oxygen consumption was measured using O2 electrodes. Protein phosphorylation was measured autoradiographically. 3. Data were collected following treatment with: (i) 8-(4-chlorophenylthio)guanosine-3',5'-monophosphate (PCPT; 10-7 or 10-5 mol/L); (ii) 8-bromo-cAMP (10-5 mol/L) followed by PCPT; (iii) beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-monophosphorothioate, SP-isomer (SP; 10-7 or 10-5 mol/L); or (iv) 8-bromo-cAMP (10-5 mol/L) followed by SP. 4. There were no significant differences between groups in baseline percentage shortening (Pcs; 4.9 +/- 0.2 vs 5.6 +/- 0.4% for control and T4 groups, respectively) and maximal rate of shortening (Rs; 64.8 +/- 5.9 vs 79.9 +/- 7.1 micro m/ s for control and T4 groups, respectively). Both SP and PCPT decreased Pcs (-43 vs-21% for control and T4 groups, respectively) and Rs (-36 vs-22% for control and T4 groups, respectively), but the effect was significantly reduced in T4 myocytes. 8-Bromo-cAMP similarly increased Pcs (28 vs 23% for control and T4 groups, respectively) and Rs (20 vs 19% for control and T4 groups, respectively). After 8-bromo-cAMP, SP and PCPT decreased Pcs (-34%) and Rs (-29%) less in the control group. However, the effects of these drugs were not altered in T4 myocytes (Pcs -24%; Rs -22%). Both PCPT and cAMP phosphorylated the same five protein bands. In T4 myocytes, these five bands were enhanced less. 5. We conclude that, in control ventricular myocytes, the cGMP-dependent protein kinase exerted major negative functional effects but, in T4-induced hypertrophic myocytes, the importance of this pathway was reduced and the interaction between cAMP and the cGMP protein kinase was diminished.

Bioactivatable, membrane-permeant analogs of cyclic nucleotides as biological tools for growth control of C6 glioma cells.

In the present study, the cAMP analogs 8-bromo-cAMP (8-Br-cAMP), N6-2'O-dibutyryl-cAMP (DBcAMP) and 8-para-chlorophenylthio-cAMP (8-CPT-cAMP), as well as the corresponding cAMP-acetoxymethyl (AM)-ester-prodrugs were tested in a HPLC study for their membrane permeability, intracellular accumulation and biotransformation. Antiproliferative activities of these compounds were studied in the rat C6 glioma cell line. Chromatographic analysis revealed that the AM-ester analogs of the cyclic nucleotides penetrate quantitatively into rat C6 glioma cells and generate high amounts of their parent cyclic nucleotides intracellularly within 60 min; however, long-term growth inhibition tested in C6 cells is only slightly enhanced with the AM-ester prodrugs of 8-Br-cAMP or DBcAMP.

Functional characterization of a novel serotonin receptor (5-HTap2) expressed in the CNS of Aplysia californica.

Serotonin has been shown to be a neuromodulator in the Aplysia californica CNS. The diversity of serotonin actions is due to the existence of several different receptor subtypes. In this study we report the cloning of a full-length cDNA, coding for a novel serotonin receptor (5-HTap2). The receptor protein bears the characteristics of G protein-coupled receptors. It shares 68% and 34% of its amino acid sequence identity with the 5-HTlym receptor from Lymnaea stagnalis and the mammalian 5-HT1A receptor, respectively. When transfected in HEK 293 cells, 5-HTap2 was negatively coupled to adenylate cyclase. Ligand binding analysis indicated that the order of potencies of various drugs for the inhibition of [3H]LSD binding was: methiothepin > metergoline > 5-CT > PAPP > 5-HT > ketanserin > NAN-190 > 8-OH-DPAT > clozapine. RT-PCR amplification of RNA isolated from different tissues indicated that this receptor is expressed in the CNS and in bag cells. The expression of 5-HTap2 restricted to the CNS suggests an important role for this receptor in the modulation of neuronal functions in Aplysia. Moreover, the high expression of 5-HTap2 in the bag cells, associated with its pharmacological profile, suggests that this receptor may be implicated in modulating the afterdischarge during the egg-laying behavior.

Cyclic nucleotide-gated channels colocalize with adenylyl cyclase in regions of restricted cAMP diffusion.

Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca(2+), cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide-gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory alpha subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide-gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22-56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide-gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide-gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.

Modeling the transformation of exogenously supplied cAMP in the perfused rat liver.

A kinetic model describing the behavior of extracellularly supplied cAMP in the perfused rat liver was derived and compared with experimental data. The model was based on the following conditions and assumptions: a) labeled cAMP is being constantly infused (step input); b) permeation of the cell membrane is an essentially irreversible step (k(in) as transfer coefficient); c) the adenine moiety of cAMP incorporates into a nucleotide pool (km1 as transformation coefficient), which cannot permeate the cell membrane; d) the adenine moiety of cAMP can be transferred from the nucleotide pool to a nucleoside + free base pool (km2 as transformation coefficient), which is able to permeate the cell membrane (k(ef) as transfer coefficient). These events were described by a series of differential equations for which an analytical solution was obtained. Total cellular incorporation of label derived from [3H]cAMP was measured in the isolated perfused rat liver. The equations of the model were fitted to these experimental data by means of a least-squares procedure. In the fitting procedure the previously determined k(in) value (0.55 ml min(-1) ml cellular space(-1)) was used. The model is able to describe the experimental data (correlation coefficient = 0.993 +/- 0.008) with km1, km2 and k(ef) values of 17.11, 0.0948 and 1.385 min(-1), respectively. Simulations revealed the following sequence of decreasing intracellular pool sizes: nucleotide pool > nucleoside + free base pool > intracellular cAMP. The intracellular cAMP concentrations correspond to only 3.2% of the extracellular ones. This low proportion explains why it was generally difficult to detect cAMP in the cell space when this compound was added to an isolated cell system. The model and the parameters determined in the present work can be used to predict intracellular cAMP concentrations in the perfused liver for specific extracellular concentrations.

Mitosis-specific phosphorylation and subcellular redistribution of the RIIalpha regulatory subunit of cAMP-dependent protein kinase.

Phosphorylation of the RII regulatory subunits of cyclic AMP-dependent protein kinases (PKAs) was examined during the HeLa cell cycle. Three RIIalpha isoforms of 51, 54, and 57 kDa were identified by RIIalpha immunodetection and labeling with 8-azido[32P]cAMP in different cell cycle phases. These isoforms were characterized as different phosphorylation states by the use of selective PKA and cyclin-directed kinase inhibitors. Whereas RIIalpha autophosphorylation by PKA caused RIIalpha to shift from 51 to 54 kDa, phosphorylation of RIIalpha by one other or a combination of several kinases activated during mitosis caused RIIalpha to shift from 51 to 57 kDa. In vivo incorporation of [32P]orthophosphate into mitotic cells and RIIalpha immunoprecipitation demonstrated that RIIalpha was hyperphosphorylated on a different site than the one phosphorylated by PKA. Deletion and mutation analysis demonstrated that the cyclin B-p34(cdc2) kinase (CDK1) phosphorylated human recombinant RIIalpha in vitro on Thr54. Whereas RIIalpha was associated with the Golgi-centrosomal region during interphase, it was dissociated from its centrosomal localization at metaphase-anaphase transition. Furthermore, particulate RIIalpha from HeLa cell extracts was solubilized following incubation with CDK1 in vitro. Our results suggest that at the onset of mitosis, CDK1 phosphorylates RIIalpha, and this may alter its subcellular localization.

Electrogenic ion transport along the human duodenum in childhood.

BACKGROUND: To find reliable sites to study the effects of different secretagogues on electrogenic ion secretion, we investigated the secretion pattern in different parts of duodenum. METHODS: Histologically normal routine intestinal biopsy specimens from children were mounted in a modified Ussing chamber. The secretory responses to prostaglandin E2 (PGE2), aminophylline, dibutyryl cyclic adenosine 5'-monophosphate (cAMP), and acetylcholine (ACh) were studied with continuous measurements of the potential difference. Tissue resistance and generated current were calculated. RESULTS: ACh induced secretion in the whole of duodenum, although the secretory response was augmented distally. PGE2 and cAMP induced significant secretion only in the distal duodenum. CONCLUSIONS: The ACh-induced, calcium-mediated, electrogenic secretion was expressed along the whole duodenum, whereas the cAMP-mediated secretion was only seen in the distal part. The fully expressed electrogenic chloride secretion was only seen at or distal to the duodenojejunal flexure. Our study shows that it is important to carefully define the localization of physiologic studies performed in the duodenum.