RESUMO
The phosphodiesterase 4 (PDE4)-cAMP pathway plays a predominant role in mediating skeletal muscle proteolysis in burn injury. The present investigations to determine the PDE4 isoform(s) involved in this action revealed that burn injury increased the expression of rat skeletal muscle PDE4B mRNA by sixfold but had little or no effect on expression of other PDE4 isoforms. These observations led us to study the effects of burn in PDE4B knockout (KO) rats. As reported by us previously, burn injury significantly increased extensor digitorum longus (EDL) muscle total and myofibrillar proteolysis in wild-type (WT) rats, but there were no significant effects on either total or myofibrillar protein breakdown in EDL muscle of PDE4B KO rats with burn injury. Moreover, burn injury increased PDE4 activity in the skeletal muscle of WT rats, but this was reduced by >80% in PDE4B KO rats. Also, burn injury decreased skeletal muscle cAMP concentration in WT rats but had no significant effects in the muscles of PDE4B KO rats. Incubation of the EDL muscle of burn-PDE4B KO rats with an inhibitor of the exchange factor directly activated by cAMP, but not with a protein kinase A inhibitor, eliminated the protective effects of PDE4B KO on EDL muscle proteolysis and increased muscle proteolysis to the same extent as in the EDL of burn-WT rats. These novel findings confirm a major role for PDE4B in skeletal muscle proteolysis in burn injury and suggest that an innovative therapy based on PDE4B-selective inhibitors could be developed to treat skeletal muscle cachexia in burn injury without the fear of causing emesis, which is associated with PDE4D inhibition.
Assuntos
Queimaduras/complicações , Caquexia/prevenção & controle , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Músculo Esquelético/enzimologia , Atrofia Muscular/prevenção & controle , Animais , Queimaduras/enzimologia , Queimaduras/genética , Caquexia/enzimologia , Caquexia/etiologia , Caquexia/genética , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Atrofia Muscular/enzimologia , Atrofia Muscular/genética , Proteólise , Ratos Sprague-Dawley , Ratos Transgênicos , Sistemas do Segundo MensageiroRESUMO
Phosphodiesterase-4 (PDE4) plays an important role in mediating memory via the control of intracellular cAMP signaling; inhibition of PDE4 enhances memory. However, development of PDE4 inhibitors as memory enhancers has been hampered by their major side effect of emesis. PDE4 has four subtypes (PDE4A-D) consisting of 25 splice variants. Mice deficient in PDE4D displayed memory enhancement in radial arm maze, water maze, and object recognition tests. These effects were mimicked by repeated treatment with rolipram in wild-type mice. In addition, similarly as rolipram-treated wild-type mice, PDE4D-deficient mice also displayed increased hippocampal neurogenesis and phosphorylated cAMP response element-binding protein (pCREB). Furthermore, microinfusion of lentiviral vectors that contained microRNAs (miRNAs) targeting long-form PDE4D isoforms into bilateral dentate gyri of the mouse hippocampus downregulated PDE4D4 and PDE4D5, enhanced memory, and increased hippocampal neurogenesis and pCREB. Finally, while rolipram and PDE4D deficiency shortened α2 adrenergic receptor-mediated anesthesia, a surrogate measure of emesis, miRNA-mediated PDE4D knock-down in the hippocampus did not. The present results suggest that PDE4D, in particular long-form PDE4D, plays a critical role in the mediation of memory and hippocampal neurogenesis, which are mediated by cAMP/CREB signaling; reduced expression of PDE4D, or at least PDE4D4 and PDE4D5, in the hippocampus enhances memory but appears not to cause emesis. These novel findings will aid in the development of PDE4 subtype- or variant-selective inhibitors for treatment of disorders involving impaired cognition, including Alzheimer's disease.
Assuntos
AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Hipocampo/citologia , Memória/fisiologia , Neurogênese/fisiologia , Interferência de RNA/fisiologia , Transdução de Sinais/fisiologia , Análise de Variância , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Bromodesoxiuridina/metabolismo , Proteína de Ligação a CREB/metabolismo , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , MicroRNAs/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/genética , Testes Neuropsicológicos , Inibidores da Fosfodiesterase 4/farmacologia , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Rolipram/farmacologia , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de TempoRESUMO
Phosphodiesterase-4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP and plays a critical role in controlling its intracellular concentration, has been implicated in depression- and anxiety-like behaviors. However, the functions of the four PDE4 subfamilies (PDE4A, PDE4B, PDE4C, and PDE4D) remain largely unknown. In animal tests sensitive to anxiolytics, antidepressants, memory enhancers, or analgesics, we examined the behavioral phenotype of mice deficient in PDE4B (PDE4B-/-). Immunoblot analysis revealed loss of PDE4B expression in the cerebral cortex and amygdala of PDE4B-/- mice. The reduction of PDE4B expression was accompanied by decreases in PDE4 activity in the brain regions of PDE4B-/- mice. Compared to PDE4B+/+ littermates, PDE4B-/- mice displayed anxiogenic-like behavior, as evidenced by decreased head-dips and time spent in head-dipping in the holeboard test, reduced transitions and time on the light side in the light-dark transition test, and decreased initial exploration and rears in the open-field test. Consistent with anxiogenic-like behavior, PDE4B-/- mice displayed increased levels of plasma corticosterone. In addition, these mice also showed a modest increase in the proliferation of neuronal cells in the hippocampal dentate gyrus. In the forced-swim test, PDE4B-/- mice exhibited decreased immobility; however, this was not supported by the results from the tail-suspension test. PDE4B-/- mice did not display changes in memory, locomotor activity, or nociceptive responses. Taken together, these results suggest that the PDE4B subfamily is involved in signaling pathways that contribute to anxiogenic-like effects on behavior.
Assuntos
Ansiedade/genética , Ansiedade/fisiopatologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Fenótipo , Análise de Variância , Animais , Ansiedade/sangue , Ansiedade/patologia , Aprendizagem da Esquiva , Comportamento Animal , Encéfalo/patologia , Encéfalo/fisiopatologia , Bromodesoxiuridina/metabolismo , Proliferação de Células , Corticosterona/sangue , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Modelos Animais de Doenças , Comportamento Exploratório , Regulação da Expressão Gênica/fisiologia , Elevação dos Membros Posteriores , Masculino , Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora , Tempo de ReaçãoRESUMO
OBJECTIVE: To assess in vivo the expression of phosphodiesterase 4 (PDE4) and its relevance to cognitive symptoms in patients with Parkinson disease (PD) using [11C]rolipram PET. METHODS: We studied 12 levodopa-treated patients with PD with no concurrent diagnosis of mild cognitive impairment or dementia. Their data were compared with those from 12 healthy controls. All participants underwent neuropsychiatric and cognitive assessment using the Cambridge Neuropsychological Test Automated Battery. Parametric images of [11C]rolipram volume of distribution (VT) values were determined with the Logan plot. RESULTS: Patients with PD performed worse than healthy controls in cognitive examinations assessing psychomotor speed, episodic memory, and spatial working memory and executive function. Patients with PD showed reductions in [11C]rolipram VT compared to healthy controls, in the caudate (28%), thalamus (23%), hypothalamus (32%), and cortex (16%). Within thalamic subregions, [11C]rolipram VT values in patients with PD were decreased by 12%-32%, with most marked decreases observed in prefrontal and temporal thalamic nuclei, whereas motor nuclei were less affected. Within the cortex, [11C]rolipram VT values in patients with PD were decreased by 11%-20%, with most marked decreases observed in posterior dorsolateral frontal cortex, medial frontal cortex, and supplementary motor area, whereas orbitofrontal cortex was less affected. Worse performance in spatial working memory correlated with lower [11C]rolipram VT values in posterior dorsolateral frontal cortex, medial frontal cortex, supplementary motor area, precentral gyrus, caudate, and prefrontal thalamic nuclei. CONCLUSIONS: Our findings demonstrate loss of PDE4 expression in the striato-thalamo-cortical circuit, which is associated with deficits of spatial working memory in patients with PD.
Assuntos
Cognição/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Doença de Parkinson/enzimologia , Doença de Parkinson/psicologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antiparkinsonianos/uso terapêutico , Mapeamento Encefálico , Radioisótopos de Carbono , Cognição/efeitos dos fármacos , Feminino , Humanos , Levodopa/uso terapêutico , Masculino , Memória de Curto Prazo/efeitos dos fármacos , Memória de Curto Prazo/fisiologia , Pessoa de Meia-Idade , Testes Neuropsicológicos , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/tratamento farmacológico , Inibidores da Fosfodiesterase 4 , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Rolipram , Memória Espacial/efeitos dos fármacos , Memória Espacial/fisiologiaRESUMO
Activation of TLR4 by lipopolysaccharide (LPS) induces both pro-inflammatory and anti-inflammatory cytokine production in macrophages. Type 4 phosphodiesterases (PDE4) are key cAMP-hydrolyzing enzymes, and PDE4 inhibitors are considered as immunosuppressors to various inflammatory responses. We demonstrate here that PDE4 inhibitors enhance the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) secretion in LPS-activated mouse peritoneal macrophages, and this response was regulated at the transcriptional level rather than an increased IL-1Ra mRNA stability. Studies with PDE4-deficient macrophages revealed that the IL-1Ra upregulation elicited by LPS alone is PKA-independent, whereas the rolipram-enhanced response was mediated by inhibition of only PDE4B, one of the three PDE4 isoforms expressed in macrophages, and it requires PKA but not Epac activity. However, both pathways activate CREB to induce IL-1Ra expression. PDE4B ablation also promoted STAT3 phosphorylation (Tyr705) to LPS stimulation, but this STAT3 activation is not entirely responsible for the IL-1Ra upregulation in PDE4B-deficient macrophages. In a model of LPS-induced sepsis, only PDE4B-deficient mice displayed an increased circulating IL-1Ra, suggesting a protective role of PDE4B inactivation in vivo. These findings demonstrate that PDE4B negatively modulates anti-inflammatory cytokine expression in innate immune cells, and selectively targeting PDE4B should retain the therapeutic benefits of nonselective PDE4 inhibitors.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Proteína Antagonista do Receptor de Interleucina 1/sangue , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-1beta/sangue , Macrófagos Peritoneais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Inibidores da Fosfodiesterase 4/farmacologia , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rolipram/farmacologia , Fator de Transcrição STAT3/metabolismo , Sepse/sangue , Sepse/patologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
RATIONALE: Phosphodiesterases (PDEs) are a super family of enzymes responsible for the halting of intracellular cyclic nucleotide signaling and may represent novel therapeutic targets for treatment of cognitive disorders. PDE4 is of considerable interest to cognitive research because it is highly expressed in the brain, particularly in the cognition-related brain regions. Recently, the functional role of PDE4B and PDE4D, two of the four PDE4 subtypes (PDE4A, B, C, and D), in behavior has begun to be identified; however, the role of PDE4A in the regulation of behavior is still unknown. OBJECTIVES: The purpose of this study was to characterize the functional role of PDE4A in behavior. METHODS: The role of PDE4A in behavior was evaluated through a battery of behavioral tests using PDE4A knockout (KO) mice; urine corticosterone levels were also measured. RESULTS: PDE4A KO mice exhibited improved memory in the step-through-passive-avoidance test. They also displayed anxiogenic-like behavior in elevated-plus maze, holeboard, light-dark transition, and novelty suppressed feeding tests. Consistent with the anxiety profile, PDE4A KO mice had elevated corticosterone levels compared with wild-type controls post-stress. Interestingly, PDE4A KO mice displayed no change in object recognition, Morris water maze, forced swim, tail suspension, and duration of anesthesia induced by co-administration of xylazine and ketamine (suggesting that PDE4A KO may not be emetic). CONCLUSIONS: These results suggest that PDE4A may be important in the regulation of emotional memory and anxiety-like behavior, but not emesis. PDE4A could possibly represent a novel therapeutic target in the future for anxiety or disorders affecting memory.
Assuntos
Ansiedade/fisiopatologia , Comportamento Animal/fisiologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/fisiologia , Animais , Corticosterona/urina , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Depressão/fisiopatologia , Emoções/fisiologia , Hipocampo/fisiopatologia , Masculino , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/fisiologia , Testes Neuropsicológicos , Isoformas de Proteínas , Memória Espacial/fisiologiaRESUMO
Phosphodiesterase-4 (PDE4) inhibitors enhance memory, increase hippocampal neurogenesis, and reverse amyloid-ß (Aß)-induced memory deficits. Here, we examined whether long-form PDE4D knockdown by lentiviral RNA construct containing a specific microRNA/miRNA-mir hairpin structure (4DmiRNA) reversed memory impairment caused by amyloid-ß1-42 (Aß42) in mice using the Morris water maze (MWM) and novelty object recognition tests. Western blotting analysis was used to assess protein levels of cAMP response element-binding protein (CREB, unphosphorylated and phosphorylated [pCREB]), brain-derived neurotrophic factor (BDNF), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and nuclear factor-κB (NF-κB) to explore the neurochemical mechanisms. Aggregated Aß42 (0.5 µg/side) bilaterally infused in dentate gyrus decreased cAMP levels (p < 0.01) and produced memory deficits in the MWM (p < 0.01) and object recognition tests (p < 0.01). Microinfusions of lentiviruses resulted in downregulated expression of PDE4D4 and 4D5 proteins and reversed Aß42-induced cAMP decline (p < 0.05) and memory deficits. Treatment also concomitantly increased pCREB (p < 0.05) and BDNF (p < 0.01) and reduced IL-1ß (p < 0.05), TNF-α (p < 0.01), and NF-κB (p65) (p < 0.05) in the hippocampus of Aß42-challenged mice. These results suggest that long-form PDE4D knockdown may offer a promising treatment for memory loss associated with Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Regulação da Expressão Gênica/fisiologia , Transtornos da Memória , Fragmentos de Peptídeos/farmacologia , Interferência de RNA/fisiologia , Animais , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Modelos Animais de Doenças , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/enzimologia , Transtornos da Memória/terapia , Camundongos , Camundongos Endogâmicos ICR , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genética , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologiaRESUMO
3'-5'-Cyclic adenosine monophosphate (cAMP) is known to be an important regulator of synaptic plasticity. The effects of cAMP are mediated through downstream effectors such as protein kinase A (PKA), Ca(2+) and cAMP-response element binding protein (CREB). The phosphodiesterase 4 (PDE4) family of enzymes, which is comprised of four genes and at least 25 protein isoforms, mediates the hydrolysis of cAMP, yet little is presently known about the contribution of specific PDE4 isoforms to synaptic plasticity and cognitive behavior. The purpose of the present studies was to determine the contribution of the PDE4B gene in mediating synaptic plasticity and cognitive behavior. Electrophysiological recordings from hippocampal slice preparations of mice deficient in the PDE4B gene (PDE4B(-/-)) showed that knockout animals displayed markedly enhanced basal postsynaptic responses to stimulation and long-term depression as compared to wild-type littermates. Interestingly, no genotypic differences were noted in long-term potentiation experiments following several different induction protocols. On the behavioral level PDE4B(-/-) mice displayed impaired reversal learning in the Morris water maze compared to wild-type littermates, but no differences in acquisition and retention of spatial memory and fear conditioning. Taken together, these results suggest that the PDE4B gene may play a role in synaptic activity and long-term depression and is involved in spatial reversal memory. Our findings support the view that various PDE4 isoforms are non-redundant and have distinct neurological roles.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Depressão Sináptica de Longo Prazo/fisiologia , Plasticidade Neuronal/fisiologia , Reversão de Aprendizagem/fisiologia , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The concept that obesity is an inflammatory state has changed our understanding of this condition and suggested that pharmacological interventions targeting inflammation may be useful strategies to improve metabolic complications of obesity. Phosphodiesterase 4 (PDE4) inhibitors exhibit profound antiinflammatory effects, but whether PDE4 inhibition suppresses obesity-induced inflammation is unknown. Among PDE4 isoforms, PDE4B is the major species mediating inflammatory responses. We therefore examined obesity-related phenotypes in mice deficient for PDE4B. Compared with wild-type littermates, PDE4B-null mice were leaner, with lower fat pad weights, smaller adipocytes, and decreased serum leptin levels on both chow and high-fat diets (HFDs). PDE4B deficiency suppressed TNF-alpha mRNA levels and macrophage infiltration in white adipose tissue in mice on HFD, but insulin sensitivity was unaltered. PDE4B-null mice on HFDs had increased locomotor activity. These results suggest a previously unappreciated role for PDE4B in the regulation of energy balance and that PDE4B inhibitors could have utility in treatment of obesity and for suppression of obesity-induced inflammation in white adipose tissue.
Assuntos
Tecido Adiposo Branco/citologia , Adiposidade/efeitos dos fármacos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Paniculite/induzido quimicamente , Adipócitos/citologia , Tecido Adiposo Branco/efeitos dos fármacos , Animais , Tamanho Celular , Gorduras na Dieta/efeitos adversos , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Leptina/sangue , Masculino , Camundongos , Atividade Motora/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In the nose, odorants are detected on the cilia of olfactory sensory neurons (OSNs), where a cAMP-mediated signaling pathway transforms odor stimulation into electrical responses. Phosphodiesterase (PDE) activity in OSN cilia has long been thought to account for rapid response termination by degrading odor-induced cAMP. Two PDEs with distinct cellular localization have been found in OSNs: PDE1C in the cilia and PDE4A throughout the cell but absent from the cilia. We disrupted both of these genes in mice and carried out electro-olfactogram analysis. Unexpectedly, eliminating PDE1C did not prolong response termination. Prolonged termination occurred only in mice that lacked both PDEs, suggesting that cAMP degradation by PDE1C in cilia is not a rate-limiting factor for response termination in wild-type mice. Pde1c(-/-) OSNs instead showed reduced sensitivity and attenuated adaptation to repeated stimulation, suggesting that PDE1C may be involved in regulating sensitivity and adaptation. Our observations provide new perspectives on the regulation of olfactory transduction.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Potenciais Evocados/fisiologia , Neurônios Receptores Olfatórios/fisiologia , Tempo de Reação/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/genética , Animais , Cálcio/farmacologia , Calmodulina/farmacologia , Simulação por Computador , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/deficiência , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Relação Dose-Resposta a Droga , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , Camundongos , Camundongos Knockout , Mucosa Olfatória/efeitos dos fármacos , Mucosa Olfatória/metabolismo , Neurônios Receptores Olfatórios/efeitos dos fármacos , Pentanóis/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/genéticaRESUMO
One of the defining properties of beta2-adrenergic receptor (beta(2)AR) signaling is the transient and rapidly reversed accumulation of cAMP. Here we have investigated the contribution of different PDE4 proteins to the generation of this transient response. To this aim, mouse embryonic fibroblasts deficient in PDE4A, PDE4B, or PDE4D were generated, and the regulation of PDE activity, the accumulation of cAMP, and CREB phosphorylation in response to isoproterenol were monitored. Ablation of PDE4D, but not PDE4A or PDE4B, had a major effect on the beta-agonist-induced PDE activation, with only a minimal increase in PDE activity being retained in PDE4D knock-out (KO) cells. Accumulation of cAMP was markedly enhanced, and the kinetics of cAMP accumulation were altered in their properties in PDE4DKO but not PDE4BKO cells. Modest effects were observed in PDE4AKO mouse embryonic fibroblasts. The return to basal levels of both cAMP accumulation and CREB phosphorylation was greatly delayed in the PDE4DKO cells, suggesting that PDE4D is critical for dissipation of the beta2AR stimulus. This effect of PDE4D ablation was in large part due to inactivation of a negative feedback mechanism consisting of the PKA-mediated activation of PDE4D in response to elevated cAMP levels, as indicated by experiments using the cAMP-dependent protein kinase inhibitors H89 and PKI. Finally, PDE4D ablation affected the kinetics of beta2AR desensitization as well as the interaction of the receptor with Galphai. These findings demonstrate that PDE4D plays a major role in shaping the beta2AR signal.