Activation of cyclic AMP pathway prevents CD34(+) cell apoptosis.

OBJECTIVE: Although cAMP is involved in a number of physiologic functions, its role in hematopoietic cell fate decision remains poorly understood. We have recently demonstrated that in CD34(+)-derived megakaryocytes, cAMP-related agents prevent apoptosis. In this study we addressed the question of whether cAMP also regulates survival of their precursors, CD34(+) cells. METHODS: Apoptosis was evaluated by fluorescence microscopy, and detection of hypodiploid or annexin V(+) cells by flow cytometry. Mitochondrial membrane potential and bcl-xL or caspase-3 expression were assessed by flow cytometry. Colony-forming units were studied by clonogenic assays in methylcellulose. RESULTS: We found that two different cAMP analogs such as Dibutiril-cAMP and sp-5,6-DCl-BIMPS (BIMPS) promoted survival of human umbilical cord-derived CD34(+) cells by suppressing apoptosis induced by either nitric oxide (NO) or serum deprivation. Involvement of PKA and PI3K pathway was demonstrated by the ability of their specific inhibitors Rp-cAMP and Wortmannin or LY294002 respectively to reverse the antiapoptotic effect of BIMPS. Treatment of CD34(+) cell with BIMPS not only restrained the bcl-xL downregulation but also suppressed the loss of mitochondrial membrane potential and caspase-3 activation induced by serum starvation. While thrombopoietin (TPO), granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF) were not able to increase cAMP levels, the antiapoptotic activity exerted by these growth factors was blocked by inhibition of the adenylate cyclase and synergized by BIMPS. Cyclic AMP analogs suppressed the decreased colony formation in cells exposed to NO or serum deprivation. CONCLUSION: Altogether, our results strongly suggest that cAMP appears to be not only a key pathway controlling CD34(+) survival, but also a mediator of the TPO-, G-CSF- and SCF-mediated cytoprotection.

Two critical periods for cAMP-dependent protein kinase activity during long-term memory consolidation in the crab Chasmagnathus.

Activation of the cAMP pathway was found to be implicated in the memory process. In the context-signal learning paradigm of the crab Chasmagnathus, the protein kinase (PKA) activator Sp-5,6-DCl-cBIMPS facilitated long-term memory (LTM) induced by spaced training while the PKA inhibitor 8-chloroadenosine-3', 5'-monophosphorothioate, Rp-isomer (Rp-8-Cl-cAMPS) produced amnesia. In the present report the effect of the PKA inhibitor on long-term retention was assessed when administered (systemic injection of 2 microg/animal) at various times after training. According to previous results obtained with a lower dose, retention is impaired when the drug is administered immediately pretraining. An effect on acquisition was ruled out considering that the drug did not affect the performance during training. On the contrary, no effect of the PKA inhibitor was found with an immediately posttraining injection and amnesia was observed only when training was shortened from 15 to 12 trials (training duration from 45 to 36 min). At 2 and 12 h posttraining Rp-8-Cl-cAMPS injection failed to impair retention, but amnesia was found when the drug was injected at 4 and 8 h after training. In order to assess a possible effect of the drug in retrieval, the PKA inhibitor was administered 15 min before testing, and no amnestic effect was observed. These results suggest that two phases of PKA activity are required during consolidation of LTM, one during training and the other between 4 and 8 h after training. The link between these two periods of PKA activation and the two phases of the transcription factor NF-kappaB activation previously found in this model, as well as the similar time course found in rodents, is discussed. An amnestic effect of the drug was not found when administered immediately before a massed training protocol that yielded an intermediate-term memory, suggesting that in this type of memory PKA activation is not required.

Effects of activation and inhibition of cAMP-dependent protein kinase on long-term habituation in the crab Chasmagnathus.

On sudden presentation of a danger stimulus, the crab Chasmagnathus elicits an escape response that habituates promptly and for a long period. We have previously reported that administration of a cAMP-permeable analog (CPT-cAMP) along with a phosphodiesterase inhibitor (IBMX) improves long-term habituation (LTH). In present experiments we studied the effect of systemic administration of the protein kinase A (PKA) activator Sp-5,6-DCl-cBIMPS and that of the PKA inhibitor Rp-8-Cl-cAMPS on LTH tested 24 h after a weak training protocol (5 trials of danger stimulus presentation) or a strong training protocol (15-30 trials), respectively. A 50 microliters pre-training injection of 75 microM Sp-5,6-DCl-cBIMPS, and to a lesser degree of 25 microM, improved retention of the habituated response but not affect short-term habituation (STH). Like pre-training injection, post-training administration of Sp-5,6-DCl-cBIMPS proved to exert a facilitatory action on retention though with 75 microM dose only. Conversely, both pre- and post-training injection of 25 microM Rp-8-Cl-cAMPS impaired LTH without affecting STH. Thus, the PKA activator Sp-5,6-DCl-cBIMPS enables a weak training to produce LTH while the PKA inhibitor Rp-8-Cl-cAMPS impairs LTH when a strong training is given. Activation of crab PKA by Sp-5,6-DCl-cBIMPS and its inhibition by Rp-8-Cl-cAMPS were assessed using an in vitro PKA activity assay. These results provide independent evidences supporting the view that PKA plays a key role in long-term memory storage in this learning paradigm.