Cyclic AMP Binding Research Articles

430. Transductional mechanisms and gene expression in melancholia

We have shown that melancholic depressives have a reduction in the exogenous phosphorylation activity of protein kinase A (PKA) relative to normal volunteers using cultured skin fibroblasts. In this study we contrasted melancholics and normals with regard to three factors: (1) The binding of [H] cyclic AMP to PKA; (2) The phosphorylation of endogenous CREB; and (3) The alteration of the expression of a CREB/CRE-dependent transfected luciferase reporter gene. Methods: Fibroblasts were cultured from drug-free subjects with DSM-IV major depression (melancholic type) and normal volunteers. [H] cyclic AMP binding (7 nM) was determined in the 48,000 3 g supernatant fraction. Cultures were incubated with 1 mM isoproterenol for 10 minutes after which CREB/CREB-P were assayed by subjecting nuclear lysates to gel electrophoresis followed by immunoblotting using polyclonal anti-CREB/CREB-P. Fibroblasts were also stably transfected with the luciferase reporter plasmid pAD neo2-C 12-BGL to evaluate any alteration in cAMP-PKA-CREB-dependent gene expression. Results: [H] cyclic AMP binding was significantly reduced in melancholics (p , 0.02): 902 6 73 vs. 631 6 51 fmol/mg protein. Isoproterenol-stimulated CREB-P and luciferase gene expression are also reduced in fibroblasts from melancholics. Both of these effects were blocked by the beta-2 antagonist ICI 118551. Conclusions: The previous finding of an abnormality of PKA activity in melancholia are now extended to suggest a reduction in CREB-P formation and an alteration in the expression of a specific CREB-CREdependent genes. We also will present newer data regarding differential expression of endogenous genes by differential display.

Increased cyclic AMP-dependent protein kinase activity in postmortem brain from patients with bipolar affective disorder.

Previous observations of reduced [3H]cyclic AMP binding in postmortem brain regions from bipolar affective disorder subjects imply cyclic AMP-dependent protein kinase function may be altered in this illness. To test this hypothesis, basal and stimulated cyclic AMP-dependent protein kinase activity was determined in cytosolic and particulate fractions of postmortem brain from bipolar disorder patients and matched controls. Maximal enzyme activity was significantly higher (104%) in temporal cortex cytosolic fractions from bipolar disorder brain compared with matched controls. In temporal cortex particulate fractions and in the cytosolic and particulate fractions of other brain regions, smaller but statistically nonsignificant increments in maximal enzyme activity were detected. Basal cyclic AMP-dependent protein kinase activity was also significantly higher (40%) in temporal cortex cytosolic fractions of bipolar disorder brain compared with controls. Estimated EC50 values for cyclic AMP activation of this kinase were significantly lower (70 and 58%, respectively) in both cytosolic and particulate fractions of temporal cortex from bipolar disorder subjects compared with controls. These findings suggest that higher cyclic AMP-dependent protein kinase activity in bipolar disorder brain may be associated with a reduction of regulatory subunits of this enzyme, reflecting a possible adaptive response of this transducing enzyme to increased cyclic AMP signaling in this disorder.

Inhibition of cyclic AMP-dependent protein kinase in the acute phase of focal cerebral ischemia in the rat

Binding of cyclic AMP to the regulatory subunit of cyclic AMP-dependent protein kinase is an essential step in cyclic AMP-mediated intracellular signal transduction. In the present study, the binding capacity of cyclic AMP-dependent protein kinase for cyclic AMP was examined by autoradiography with local cerebral blood flow in focal cerebral ischemia in the rat, which was induced by occlusion of the middle cerebral artery using the intraluminal suture method. The binding capacity of cyclic AMP-dependent protein kinase and local cerebral blood flow were assessed by the in vitro [ 3H]cyclic AMP binding and the [ 14C]iodoantipyrine methods, respectively. At 3 h of occlusion, a significant reduction in the binding of cyclic AMP-dependent protein kinase to cyclic AMP was already noted in the lateral region of the caudate–putamen and the parietal cortex. Between three and five hours of occlusion, the area with reduced cyclic AMP binding was significantly expanded to the peri-ischemic regions including the frontal cortex and the medial region of the caudate–putamen. The threshold in local cerebral blood flow for reduced cyclic AMP binding was clearly noted at 5 h of ischemia, and was 45 ml/100 g per min in the cerebral cortices, and 38 ml/100 g per min in the caudate–putamen, respectively. No threshold was noted at 3 h of ischemia, since cyclic AMP binding showed a large variation ranging from reduced to normal values even when local cerebral blood flow was below 20 ml/100 g per min. Recirculation for 3.5 h following 1.5 h of ischemia restored the normal cyclic AMP binding in the cerebral cortices, but failed to normalize cyclic AMP binding in the caudate–putamen despite good recovery of local cerebral blood flow. Western blot analysis suggested that this reduction in cyclic AMP binding was not due to loss or degradation of the subunit protein of cyclic AMP-dependent protein kinase, and may therefore have resulted from conformational changes in the protein. A significant increase in cyclic AMP binding was noted after recirculation in the non-ischemic regions such as the frontal and the cingulate cortices on the occluded side and in the contralateral cortices. These data indicate that cyclic AMP-mediated signal transduction in the brain tissue may be very susceptible to ischemic stress, and the region of disrupted signal transduction may expand progressively from the ischemic core to peri-ischemic regions in the acute phase of ischemia. Such impairment of signal transduction may not be restored in the caudate–putamen even when cerebral circulation is fully recovered after short-term ischemia, suggesting that a regional vulnerability to ischemic stress may also exist in cyclic AMP-mediated signal transduction. A significant increase in cyclic AMP binding after recirculation in regions outside of ischemic area may be closely related with the protective mechanisms of brain tissue, since cyclic AMP has been reported to exert various neuroprotective actions.

Time-course changes in rat cerebral cortex subcellular distribution of the cyclic-AMP binding after treatment with selective serotonin reuptake inhibitors.

Pharmacological investigations have suggested the involvement of the cAMP transduction pathway in the action of antidepressant drugs and in the pathophysiology of mood disorders. We have extended these studies to determine the time-related effects of two selective serotonin reuptake inhibitors, fluvoxamine (15 mg/kg) and paroxetine (5 mg/kg), on the cAMP-binding in rat cerebral cortex, after short and long-term treatments. Photoaffinity labelling experiments with 8-N(3)-[(32)P]cAMP were carried out in cerebrocortical soluble (S1 or S2) and microtubule fractions. In our conditions, both SSRIs administered for 5 days were unable to affect the cAMP-binding in S1, S2, and in microtubule fractions. After 12 days of treatment, paroxetine and fluvoxamine significantly enhanced the cAMP-binding to the 54 kDa protein, corresponding to the type II regulatory subunit of PKA (RII), in the S1 and microtubule fractions. Any modification in respect to controls was observed in S2, the soluble fraction devoid of microtubules. After 21 days of treatment no changes were observed in the soluble S1 fraction and in microtubules, but the cAMP-binding to the RII subunit was found to be significantly higher in the S2 fraction. The high concentration of RII, demonstrated first in microtubules (12 days) and then in the cytosol (21 days), could be the result of a time-related effect of SSRIs on PKA and its translocation from microtubule compartment to the cytosol. The present findings seem to demonstrate the capacity of SSRIs to modulate the subcellular distribution of PKA and support the involvement of the cAMP pathway in the mechanism of action of these drugs.

Acute ischemic vulnerability of PKA in the dendritic subfields of the hippocampus CA1

Alterations of [3H]cyclic AMP (cAMP) binding, an indicator of the binding activity of particulate cyclic AMP-dependent protein kinase (PKA), were examined after 15 and 30 min of ischemia in the gerbil brain. Severe hemispheric cerebral ischemia was induced by occluding the right common carotid artery. Significant reductions in cAMP binding were noted only in the dendritic subfields of the hippocampus CA1 such as the strata oriens, radiatum and lacunosum-moleculare, on the ischemic side after 15 min of ischemia. After 30 min ischemia cAMP binding was significantly decreased not only in each dendritic subfield of the hippocampus CA1, but also in the layer of pyramidal cell bodies (stratum pyramidale) on the occluded side; other brain regions such as the hippocampus CA3, dentate gyrus and cerebral cortices revealed no significant changes in cAMP binding. These findings suggest that derangement of PKA may begin in the dendritic subfields of the hippocampus CA1 after as little as 15 min of severe ischemia, and proceed centrally to the neuronal cell bodies of the hippocampus CA1.

Clathrin heavy chain is required for spore cell but not stalk cell differentiation in Dictyostelium discoideum.

Previous studies of a clathrin-minus Dictyostelium cell line revealed important roles for clathrin heavy chain (clathrin) in endocytosis, secretion of lysosomal hydrolases and osmoregulation. In this paper, we examine the contribution of clathrin-mediated membrane traffic to development in Dictyostelium discoideum. Clathrin-minus cells were delayed in early development. When exposed to starvation conditions, clathrin-minus cells streamed and aggregated more slowly than wild-type cells. Although clathrin-minus cells displayed only 40% the level of extracellular cyclic AMP binding normally found in wild-type cells, they responded chemotactically to extracellular cyclic AMP. Clathrin-minus cells down-regulated cyclic AMP receptors, but only to half the extent of wild-type cells. We found that the extent of development of clathrin-minus cells was variable and influenced by environmental conditions. Although the mutant cells always progressed beyond the tipped mound stage, the final structure varied from a finger-like projection to a short, irregular fruiting body. Microscopic examination of these terminal structures revealed the presence of intact stalks but a complete absence of spores. Clathrin-minus cells expressed prestalk (ecmA and ecmB) and prespore (psA and cotB) genes normally, but were blocked in expression of the sporulation gene spiA. Using clathrin-minus cells that had been transformed with various promoter-lacZ reporter constructs, we saw only partial sorting of clathrin-minus prestalk and prespore cells. Even when mixed with wild-type cells, clathrin-minus cells failed to sort correctly and never constructed functional spores. These results suggest three roles for clathrin during Dictyostelium development. First, clathrin increases the efficiency of early development. Second, clathrin enables proper and efficient patterning of prestalk and prespore cells during culmination. Third, clathrin is essential for differentiation of mature spore cells.

Oxidative Modulation of Cyclic AMP-Dependent Protein Kinase in Human Fibroblasts: Possible Role in Psoriasis

Previous studies have established that cyclic AMP-dependent protein kinase (PKA) activity, as well as 8-azido-[32P]-cAMP binding to the RI and RII regulatory subunits, are decreased in cells from psoriatic patients compared to cells from normal patients. Here we show that the exposure of normal human dermal fibroblasts in culture to hydrogen peroxide and to oxygen free-radical generating systems decreased PKA activity, as well as cyclic AMP binding to the RI and RII regulatory subunits, to levels similar to those observed with psoriatic fibroblasts. Likewise, treatment of normal cytosolic preparations of PKA, as well as purified bovine PKA II, in vitro with free radical generating systems also resulted in decreased PKA activity and 8-azido [32P]-cAMP binding to the RI and RII regulatory subunits. Further, treatment of psoriatic fibroblasts with free radical scavenging agents such as vitamins E and C, and mannitol, and also with superoxide dismutase, restored the ability of RI and RII to bind 8-azido-[32P]-cAMP toward normal levels. Western blot analysis showed that the protein levels of the RI and RII subunits are similar in normal and psoriatic fibroblasts, and that the amounts of RI and RII are not altered by treatment of the cells with free radical-generating systems. These results suggest that oxidative modification may serve as a mechanism to alter PKA activity in human cells, and that an altered oxidative state may be involved in mediating the decrease in PKA activity and cyclic AMP binding noted in cells from psoriatic patients.Copyright © 1997 Elsevier Science Inc.

Reduced [3H]cyclic AMP binding in postmortem brain from subjects with bipolar affective disorder.

Findings of increased Gs alpha levels and forskolin-stimulated adenylyl cyclase activity in selective cerebral cortical postmortem brain regions in bipolar affective disorder (BD) implicate increased cyclic AMP (cAMP)-mediated signaling in this illness. Accumulating evidence suggests that intracellular levels of cAMP modulate the abundance and disposition of the regulatory subunits of cAMP-dependent protein kinase (cAMP-dPK). Thus, in the present study, we tested further whether hyperfunctional Gs alpha-linked cAMP signaling occurs in BD by determining [3H]cAMP binding, a measure of the levels of regulatory subunits of cAMP-dPK, in cytosolic and membrane fractions from discrete brain regions of postmortem BD brain. Specific [3H]cAMP (5 nM) binding was determined in autopsied brain obtained from 10 patients with DSM-III-R diagnoses of BD compared with age- and postmortem delay-matched controls. [3H]cAMP binding was significantly reduced across all brain regions in cytosolic fractions of BD frontal (-22%), temporal (-23%), occipital (-22%) and parietal (-15%) cortex, cerebellum (-36%), and thalamus (-13%) compared with controls, but there were no differences in [3H]cAMP binding in the membrane fractions from these same regions. These results suggest that changes occur in the cAMP-dPK regulatory subunits in BD brain, possibly resulting from increased cAMP signaling. The possibility that antemortem lithium and/or other mood stabilizer treatment may contribute to the above changes, however, cannot be ruled out.

Arginine 210 Is Not a Critical Residue for the Allosteric Interactions Mediated by Binding of Cyclic AMP to Site A of Regulatory (RIα) Subunit of Cyclic AMP-dependent Protein Kinase

The guanidinium groups of conserved arginines in the two intrachain cAMP-binding sites of regulatory (R) subunit of cAMP-dependent protein kinase have been implicated in the allosteric interactions by which cAMP binding leads to kinase activation. We have investigated the functional role of Arg-210, the conserved arginine in site A of murine type Ialpha R subunit, by analyzing the effects of nine different substitutions at this residue on cAMP binding and allosteric properties of bacterially expressed RIalpha subunits. All substitutions reduced the cAMP binding affinity of site A, but the magnitude of reduction varied from several hundredfold to 10(6)-fold. The differential effects of the different substitutions could not easily be rationalized by interactions with cAMP and might, in part, reflect interactions with other residues in the unoccupied cAMP-binding pocket. None of the Arg-210 substitutions appeared to disrupt the allosteric interaction by which occupation of site A slows dissociation of cAMP from site B, although the effect was difficult to elicit in full with mutations that had strong effects on cAMP binding. The two weakest substitutions, Arg-210 --> Ile and Arg-210 --> Thr, could be shown to have essentially no effect on the allosteric interaction by which occupation of site A reduces the affinity of R subunit for the catalytic subunit. The weaker mutations had a smaller effect on kinase activation by the suboptimal activator Rp-adenosine cyclic 3',5'-phosphorothioate than by cAMP, suggesting that the analog largely bypasses interactions with the guanidinium group of Arg-210.

Flow Threshold for Reduction of Cyclic AMP Binding in the Hippocampus CA1 and other Brain Regions during Stroke Development in Gerbils

The flow threshold for alterations of the in vitro [3H]cyclic AMP (cAMP) binding, an indicator of the total amount of particulate cAMP-dependent protein kinase, was evaluated in the gerbil brain after 30 min, 2 h, and 6 h of unilateral common carotid artery occlusion, respectively. The autoradiographic method developed in our laboratory enabled us to measure the [3H]cAMP binding and local CBF in each region of the same brain. The ischemic flow thresholds for reduction of the cAMP binding in the hippocampus CA1 were 18, 34, and 49 ml 100 g-1 min-1 after 30-min, 2-h, and 6-h ischemia, respectively. These values were higher than those in other regions such as the hippocampus CA, and temporal cerebral cortex in each duration of ischemia. These findings indicate that (a) the ischemic flow threshold for perturbation of the cAMP system may be higher in the hippocampus CA1 than in other brain regions, suggesting that the hippocampus CA1 could be especially vulnerable to acute ischemic stress; and (b) the level of the aforementioned threshold may increase progressively during the time course of ischemia in particular regions such as the hippocampus CA1 and CA3, suggesting that the duration of ischemia exerts a definite influence on the viability of the ischemic neuronal cells in these regions.

Characterization of protein kinase and phosphatase systems in chick ciliary ganglion.

The aim of the present study was to characterize the second messenger activated protein kinase and phosphatase systems in chick ciliary ganglion using biochemical and immunochemical techniques. Using synthetic peptide substrates cyclic-AMP-, cyclic-GMP-, Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinase activities were detected in homogenates of ciliary ganglion dissected from 15-16-day-old embryos. Autophosphorylation of the alpha and beta subunits of Ca2+/calmodulin-dependent protein kinase II in the presence of Ca2+/calmodulin or 5 mM ZnSO4 was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and autoradiography. Protein kinase C was shown to be present using a monoclonal antibody. Two cyclic-AMP binding proteins whose molecular weights corresponded to the regulatory subunits of cyclic AMP-dependent protein kinase (RI and RII) were detected in ciliary ganglia using 8-azido-cyclic-AMP. The most heavily labelled band following incubation with [gamma-32P]ATP under most conditions had an apparent molecular weight of 65,000 which corresponds to the chicken form of myristoylated alanine-rich C kinase substrate, a known substrate of protein kinase C. Another substrate for protein kinase C was a 45,000 molecular weight protein which was tentatively identified as neuromodulin (B-50/GAP-43). Although no endogenous substrate proteins for cyclic-GMP-dependent protein kinase were detected, protein kinase A strongly labelled a 40,000 molecular weight protein. Using 32P(i)-labelled glycogen phosphorylase, protein phosphatases 1 and 2A were identified in ciliary ganglia homogenates at levels which were indistinguishable from forebrain at the same age. The major endogenous protein substrates in ciliary ganglion homogenates from 15-16-day-old embryos were also labelled to a similar extent in homogenates of ciliary ganglia from newly hatched chickens. Intact ciliary ganglia remained viable for several hours after dissection and, after incubation with 32P(i), responded to phorbol ester stimulation by an increased endogenous phosphorylation of several proteins, but especially myristoylated alanine-rich C kinase substrate. These results represent the first systematic characterization of the protein phosphorylation systems in chicken ciliary ganglion and provide a basis for future studies on the biochemical mechanisms responsible for regulating synaptic transmission in this tissue.

β-adrenergic mitogenic signal transduction in peripheral lung adenocarcinoma: Implications for individuals with preexisting chronic lung disease

Peripheral adenocarcinoma (PAC) of the lung has increased dramatically over the last 20 years and is today the leading histological type of lung cancer in smokers and nonsmokers in industrialized countries. There is no apparent explanation for the steep rise in the number of individuals developing this cancer type. Using assays for the assessment of cell proliferation, receptor binding, and production of cyclic AMP (cAMP), we have identified a beta-adrenergic receptor-mediated mitogenic pathway, which activates cAMP down-stream, in cell lines derived from human peripheral adenocarcinomas that express features of Clara cells. Agonists of beta-adrenergic receptors strongly stimulated cell proliferation, whereas antagonists of this receptor and its associated second messenger, cAMP, were potent inhibitors of this effect. Agonists of beta-adrenergic receptors are the active ingredients of many decongestants and bronchodilators, and such medications are, therefore, likely to stimulate this pathway in vivo. Patients suffering from chronic upper and lower respiratory tract diseases and treated with such medications over many years may, therefore, be at a higher risk than the average population to develop PAC, particularly when simultaneously exposed to carcinogenic environmental factors such as smoking. Because the incidence of chronic respiratory tract diseases has risen in industrialized countries during the same time frame as PAC, a potential etiological link between the therapy of such nonneoplastic diseases with beta-adrenergic agonists and the risk for PAC should be investigated.

The cyclic adenosine 3′,5′ monophosphate-dependent protein kinase system in breast cancer

Summary Components of the cyclic adenosine 3′,5′ monophosphate (AMP) dependent protein kinase sys- tem have been measured in primary tumours from 55 patients with invasive breast cancer. There were highly significant positive correlations between levels of regulatory subunits as measured by cyclic AMP binding proteins and catalytic kinase activity (both basal and cyclic AMP-activated). This suggests that, in general, increased expression of binding proteins is accompanied by a corresponding elevated level of catalytic activity. Nevertheless, individual tumours could have substantially different proportions of regulatory and catalytic activities. Whether such differences influence tumour behaviour remains to be determined.

Growth inhibition by 8-chloro cyclic AMP of human HT29 colorectal and ZR-75-1 breast carcinoma xenografts is associated with selective modulation of protein kinase A isoenzymes

Significant dose-related inhibition of growth of HT29 human colorectal cancer xenografts and ZR-75-1 breast cancer xenografts in immune-suppressed mice was induced by the cyclic AMP analogue, 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cyclic AMP) when given by alzet mini-pumps over a 7-day period at doses of either 50 or 100 mg/kg/day. Levels and types of cyclic AMP binding proteins were measured by ligand binding and photoaffinity labelling, respectively, in tumours harvested at the end of the treatment period. Compared with levels in tumours from control animals, values of tumour cyclic AMP binding proteins from treated animals were significantly reduced. These effects were associated with an apparent modulation of the types of cyclic AMP binding proteins, 8-Cl-cyclic AMP-treated xenografts displaying a reduced ratio of RI/RII isoforms compared with untreated control tumours.

Biochemical changes, early brain growth suppression and impaired detour learning in nicotine-treated chicks

Fetal growth suppression associated with chronic maternal intake of cigarette smoke is frequently observed in humans and studies using animal models suggest that in utero nicotine exposure is an important component of this growth suppression. The developing fetal central nervous system (CNS) is sensitive to the growth inhibitory effect of nicotine and morphological as well as functional CNS deficits may result from fetal nicotine exposure. The studies presented here show that nicotine exposure during early embryonic development ultimately inhibits the ability of 7–11 day old chicks to learn a detour task. The brain growth suppression caused by nicotine is paralleled by a failure of the early embryo brain to express the normal developmental increase in ornithine decarboxylase (ODC) activity. This biochemical change may be germane to the mechanism of nicotine-induced growth inhibition and/or nicotine-induced behavioral changes because the appropriate expression of ODC activity is essential to normal growth and differentiation in the fetal CNS. In the chick embryo, nicotine exposure alters several important signaling pathways that regulate ODC expression. For example, nicotine exposure lowers embryonic brain glucose levels and causes significant decreases in whole brain cyclic adenosine 3′,5′-monophosphate (cyclic AMP) levels and in cyclic AMP binding proteins (protein kinase-A regulatory activity). Also, in cultured chick cells, nicotine inhibits the ability of a potent mitogen (insulin) to induce ODC activity, but, paradoxically, in ovo nicotine exposure increased insulin binding and stimulated insulin receptor autophosphorylation in brain membranes.

Age-dependent changes in second messenger and rolipram receptor systems in the gerbil brain.

Age-related alterations in binding sites of major second messengers and a selective adenosine 3',5'-cyclic monophosphate (cyclic-AMP) phosphodiesterase (PDE) in the gerbil brain were analysed by receptor autoradiography. [3H]Phorbol 12,13-dibutyrate (PDBu), [3H]inositol 1,4,5-trisphosphate (IP3), [3H]forskolin, [3H]cyclic-AMP, and [3H]rolipram were used to label protein kinase C (PKC), IP3 receptor, adenylate cyclase, cyclic-AMP dependent protein kinase (PKA), and Ca2+/calmodulin-independent cyclic-AMP PDE, respectively. In middle-aged gerbils (16 months old), [3H]PDBu binding was significantly reduced in the hippocampal CA1 sector, thalamus, substantia nigra, and cerebellum, compared with young animals (1 month old). [3H]IP3 binding revealed significant elevations in the nucleus accumbens, hippocampal CA1 sector, dentate gyrus, and a significant reduction in cerebellum of middle-aged gerbils. [3H]Forskolin binding in middle-aged animals was significantly increased in the nucleus accumbens and hilus of dentate gyrus, but was diminished in the substantia nigra and cerebellum. On the other hand, in middle-aged animals, [3H]cyclic-AMP binding revealed a significant elevation only in the hippocampal CA3 sector, whereas [3H]rolipram binding showed a significant reduction in the thalamus and cerebellum. Thus, the age-related alteration in these binding sites showed different patterns among various brain regions in middle-aged gerbils indicating that the binding sites of PKC, IP3, and adenylate cyclase are more markedly affected by aging than those of PKA and cyclic-AMP PDE and that the hippocampus and cerebellum are more susceptible to these aging processes than other brain regions. The findings suggest that intracellular signal transduction is affected at an early stage of senescence and this may lead to neurological deficits.

Bacterial expression of Chinese hamster regulatory type-I and catalytic subunits of cyclic AMP-dependent protein kinase and mutational analysis of the type-I regulatory subunit

The type-I regulatory subunit (RI) of the cyclic AMP-dependent protein kinase (PKA) from Chinese hamster ovary (CHO) cells has been cloned and expressed in a strain of BL21(DE3) Escherichia coli lacking adenylate cyclase [BL21(DE3)/delta cya]. RI expressed in this bacterial system free of cyclic AMP is soluble and can reconstitute functional PKA. Recombinant CHO C alpha is predominantly insoluble with some active soluble protein. C beta is entirely insoluble and inactive. Soluble recombinant RI and soluble recombinant C alpha can associate in vitro and be activated by cyclic AMP. Six site-directed mutations of RI were generated to study the interaction of cyclic AMP with RI and RI-C alpha subunit interactions. Four cyclic AMP-binding-site point mutants were generated [W261R (tryptophan to arginine at position 261), a novel mutation in site A; V376G, a novel mutation in site B; G200E (site A), and Y370F (site B), previously described in bovine RI were introduced into the CHO RI for comparison purposes]. Mutants W261R, Y370F, and G200E demonstrated decreased 8-N3-[3H]cyclic AMP binding as well as 5-fold reduced affinity for [3H]cyclic AMP, with threefold increased EC50 values for cyclic AMP activation of kinase activity from reconstituted mutant holoenzymes. The mutation at V376G did not alter cyclic AMP binding or activation by cyclic AMP of mutant holoenzyme. A truncation mutant, G200Stop, which lacks both cyclic AMP-binding sites, did not bind cyclic AMP but can inhibit C alpha subunit activity. A novel mutation outside the cyclic AMP-binding regions of RI (V89A) weakened the interaction with C alpha indicated by a 7-fold lower EC50 for mutant holoenzyme activation by cyclic AMP.

Patterns of cyclic AMP binding in normal human breast.

Cyclic AMP binding proteins have been measured in normal breast tissue, in a variety of physiological conditions including resting state (69 cases), pregnancy (15 cases), lactation (4 cases), post-lactational involution (6 cases), and prolonged involution (10 cases). Levels varied greatly within the groups but median values were elevated in pregnancy, lactation, and post-lactational involution as compared with the resting and prolonged involutionary states. The proportion of different types of cyclic AMP binding proteins also differed between groups. Resting breast showed approximately equal amounts of type I and II whereas pregnant and postlactational involuting breast tissue had an increased proportion of type I binding, and the prolonged involutionary state tended to be associated with low type I. Within the subgroup of nulliparous resting breasts, the proportion of type I cyclic AMP binding protein was significantly elevated during the second half of the menstrual cycle compared with the first (p < 0.05). There were also significant positive associations between the level of epithelial proliferation and both total (p < 0.015) and type I (p < 0.002) cyclic AMP binding. This represents the strongest of association thus far reported between signalling systems and proliferation in the normal breast.

Changes of [3H]cyclic adenosine monophosphate binding in the gerbil brain following transient cerebral ischemia: an autoradiographic study and investigation of the effects of vinconate and pentobarbital.

We studied the alterations in binding of cyclic AMP as an indicator of particulate cyclic AMP-dependent protein kinase binding activity following transient cerebral ischemia in Mongolian gerbils and examined the effects of vinconate and pentobarbital against alterations in the binding. Animals were allowed to survive for 5 h and 7 days after 10 min of cerebral ischemia induced by bilateral occlusion of common carotid arteries. [3H]Cyclic AMP binding was significantly reduced in the hippocampus 5 h after ischemia, whereas the striatum showed no significant change in the binding. Seven days after ischemia, a severe reduction of [3H]cyclic AMP binding was noted in the dorsolateral striatum, hippocampal CA1 and CA3 sectors, and dentate gyrus. Intraperitoneal administration of vinconate (100 or 300 mg/kg) showed a significant elevation of [3H]cyclic AMP binding in the striatum, stratum pyramidale of hippocampal CA1 and CA3 sectors, and dentate gyrus 5 h after ischemia. By contrast, the intraperitoneal administration of pentobarbital (40 mg/kg) showed no significant alteration of [3H]cyclic AMP binding in most of these regions. However, vinconate and pentobarbital prevented a significant reduction of [3H]cyclic AMP binding in the dorsolateral striatum and stratum pyramidale of hippocampal CA3 sector 7 days after ischemia, although both drugs failed to prevent damage to the hippocampal CA1 sector. These results suggest that alteration in cyclic AMP binding may not be a major factor in causing ischemic neuronal damage.

Cyclic AMP binding to the amphibian oocyte plasma membrane: possible interrealtionship between meiotic arrest and membrane fluidity

Cyclic AMP, which maintains the vertebrate oocyte in prophase arrest under physiological conditions, exhibits specific and saturable binding to the cytoplasmic face of the prophase-arrested Rana pipiens oocyte membrane. Scatchard type analyses of [ 3H]cAMP binding to isolated plasma membranes indicate a single class of binding sites with a K d = 19.3 ± 7.0 nM at cAMP concentrations below 10 −6 M and additional low affinity site(s) and/or non-specific binding at concentrations above 10 −6 M. Photoaffinity labeling of prophase oocyte plasma membranes with [ 32P]-8-N 3cAMP demonstrates cAMP/cGMP-displacable binding of 8-N 3[ 32P]cAMP to a 100–110 kDa peptide doublet. Plasma membrane fluidity was monitored by electron spin resonance in isolated plasma-vitelline membranes using a 5-doxyl stearic acid probe. Exogenous dibutryl cAMP (dbcAMP) produces an increase in membrane fluidity within minutes and blocks and/or reverses the progesterone-induced decrease in plasma membrane fluidity. The dbcAMP concentration that produced half-maximal fluidity increase (10 μM) corresponds to the half-maximal inhibiting dose of dbcAMP for progesterone induction of meiosis. Cholera toxin, which elevates intracellular cAMP and blocks meiosis, also increases membrane fluidity and inhibits progesterone-induced decrease in membrane fluidity. Elevated levels of intracellular cAMP thus appear to maintain meiotic arrest by binding to specific plasma membrane site(s) and maintaining the plasma membrane in a relatively fluid state. The progesterone-induced fall in intracellular cAMP first reported in Rana thus appears to be responsible for the progesterone-induced increase in membrane fluidity and further suggests that the change in membrane order is essential for the resumption of the meiotic divisions.