Catechol-O-methyltransferase inhibition increases pain sensitivity through activation of both β2- and β3-adrenergic receptors

Catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines, has recently been implicated in the modulation of pain. Our group demonstrated that human genetic variants of COMT are predictive for the development of Temporomandibular Joint Disorder (TMJD) and are associated with heightened experimental pain sensitivity (Diatchenko et al. 2005). Variants associated with heightened pain sensitivity produce lower COMT activity. Here we report the mechanisms underlying COMT-dependent pain sensitivity. To characterize the means whereby elevated catecholamine levels, resulting from reduced COMT activity, modulate heightened pain sensitivity, we administered a COMT inhibitor to rats and measured behavioral responsiveness to mechanical and thermal stimuli. We show that depressed COMT activity results in enhanced mechanical and thermal pain sensitivity. This phenomenon is completely blocked by the nonselective β-adrenergic antagonist propranolol or by the combined administration of selective β2- and β3-adrenergic antagonists, while administration of β1-adrenergic, α-adrenergic, or dopaminergic receptor antagonists fail to alter COMT-dependent pain sensitivity. These data provide the first direct evidence that low COMT activity leads to increased pain sensitivity via a β2/3-adrenergic mechanism. These findings are of considerable clinical importance, suggesting that pain conditions resulting from low COMT activity and/or elevated catecholamine levels can be treated with pharmacological agents that block both β2- and β3-adrenergic receptors

Signaling pathways mediating β3-adrenergic receptor-induced production of interleukin-6 in adipocytes

The β3-adrenergic receptor (β3AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of β3AR activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the β3AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific β3AR agonist {"type":"entrez-nucleotide","attrs":{"text":"CL316243","term_id":"44896132","term_text":"CL316243"}}CL316243 in adipocytes, cells that exhibit the highest expression of β3ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the Gs-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-κB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block β3AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the β3AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the β3AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways

Synonymous Genes Explore Different Evolutionary Landscapes

The evolutionary potential of a gene is constrained not only by the amino acid sequence of its product, but by its DNA sequence as well. The topology of the genetic code is such that half of the amino acids exhibit synonymous codons that can reach different subsets of amino acids from each other through single mutation. Thus, synonymous DNA sequences should access different regions of the protein sequence space through a limited number of mutations, and this may deeply influence the evolution of natural proteins. Here, we demonstrate that this feature can be of value for manipulating protein evolvability. We designed an algorithm that, starting from an input gene, constructs a synonymous sequence that systematically includes the codons with the most different evolutionary perspectives; i.e., codons that maximize accessibility to amino acids previously unreachable from the template by point mutation. A synonymous version of a bacterial antibiotic resistance gene was computed and synthesized. When concurrently submitted to identical directed evolution protocols, both the wild type and the recoded sequence led to the isolation of specific, advantageous phenotypic variants. Simulations based on a mutation isolated only from the synthetic gene libraries were conducted to assess the impact of sub-functional selective constraints, such as codon usage, on natural adaptation. Our data demonstrate that rational design of synonymous synthetic genes stands as an affordable improvement to any directed evolution protocol. We show that using two synonymous DNA sequences improves the overall yield of the procedure by increasing the diversity of mutants generated. These results provide conclusive evidence that synonymous coding sequences do experience different areas of the corresponding protein adaptive landscape, and that a sequence's codon usage effectively constrains the evolution of the encoded protein

Signaling pathways mediating β3-adrenergic receptor-induced production of interleukin-6 in adipocytes

The β3-adrenergic receptor (β3AR) is an essential regulator of metabolic and endocrine functions. A major cellular and clinically significant consequence of β3AR activation is the substantial elevation in interleukin-6 (IL-6) levels. Although the β3AR-dependent regulation of IL-6 expression is well established, the cellular pathways underlying this regulation have not been characterized. Using a novel method of homogenous reporters, we assessed the pattern of activation of 43 transcription factors in response to the specific β3AR agonist {"type":"entrez-nucleotide","attrs":{"text":"CL316243","term_id":"44896132","term_text":"CL316243"}}CL316243 in adipocytes, cells that exhibit the highest expression of β3ARs. We observed a unique and robust activation of the CRE-response element, suggesting that IL-6 transcription is regulated via the Gs-protein/cAMP/protein kinase A (PKA) but not nuclear factor kappa B (NF-κB) pathway. However, pretreatment of adipocytes with pharmacologic inhibitors of PKA pathway failed to block β3AR-mediated IL-6 up-regulation. Additionally, stimulation of adipocytes with the exchange protein directly activated by cAMP (Epac) agonist did not induce IL-6 expression. Instead, the β3AR-mediated transcription of IL-6 required activation of both the p38 and PKC pathways. Western blot analysis further showed that transcription factors CREB and ATF-2 but not ATF-1 were activated in a p38- and PKC-dependent manner. Collectively, our results suggest that while stimulation of the β3AR leads to a specific activation of CRE-dependent transcription, there are several independent cellular pathways that converge at the level of CRE-response element activation, and in the case of IL-6 this activation is mediated by p38 and PKC but not PKA pathways