Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics.

Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iß isoforms are present in preadipocytes, only PKG-Iß isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems.

cGMP and cGMP-dependent protein kinase I pathway in dorsal root ganglia contributes to bone cancer pain in rats.

STUDY DESIGN: A prospective, randomized experimental research. OBJECTIVE: To demonstrate the role of cGMP (cyclic guanosine monophosphate)-cGKI (cGMP-dependent protein kinase I) pathway in dorsal root ganglia (DRG) in bone cancer pain. SUMMARY OF BACKGROUND DATA: Treating bone cancer pain continues to possess a major clinical challenge because the specific cellular and molecular mechanisms underlying bone cancer pain remain elusive. cGMP and cGMP-dependent protein kinases pathway in DRG plays important role in nerve injury-induced hyperexcitability of DRG neurons, as well as neuropathic pain, however, whether this pathway participates in bone cancer pain is unknown. METHODS: The rat model of bone cancer pain was produced by intramedullary injection of rat breast cancer cells (Walker 256) into right tibia. Thermal hyperalgesia and mechanical allodynia were measured before and after administration of inhibitor of cGMP-cGKs pathway (Rp-8-pCPT-cGMPS). Immunofluorescence and reverse transcription-polymerase chain reaction were used to reflect expression of cGKI in DRG neurons, whereas the concentration of cGMP in DRG was tested using enzyme-linked immunosorbent assay method. Whole-cell patch clamp was used to record the hyperexcitability of small neurons in DRG with or without cGKs inhibitor after tumor cell implantation (TCI). RESULTS: TCI treatment significantly increased the concentration of cGMP in DRG and activity of cGKs in DRG and the spinal cord. TCI treatment also induced upregulation of cGKI messenger ribonucleic acid and protein in DRG, as well as enhanced hyperexcitability in DRG neurons. Spinal administration of Rp-8-pCPT-cGMPS, cGMP-cGKs inhibitor, significantly suppressed TCI-induced activation of cGMP-cGKI signaling, and hyperexcitability of DRG neurons. Meanwhile, in vivo intrathecal delivery of the Rp-8-pCPT-cGMPS significantly prevented and suppressed TCI-induced hyperalgesia and allodynia. CONCLUSION: From these results, we confirm that TCI treatment activates cGMP-cGKI signaling pathway and continuing activation of this pathway in DRG is required for hyperalgesia and/or hyperalgesia and allodynia after TCI treatment. LEVEL OF EVIDENCE: N/A.

In vivo blockade of α1-adrenergic receptors mitigates stress-disturbed cAMP and cGMP signaling in Leydig cells.

The molecular mechanism of stress-associated reproductive dysfunction is complex and largely unknown. This study was designed to systematically analyze molecular effects of systemic in vivo blockade of α1-adrenergic receptors (α1-ADRs) on stress-induced disturbance of cAMP/cGMP signaling in testosterone-producing Leydig cells using the following parameters (i) level of circulating stress hormones, LH and testosterone; (ii) level of main molecular markers of Leydig cell functionality (testosterone, Insl3, cAMP); (iii) expression of cAMP signaling (cAMP 'producers'/'effectors'/'removers') and (iv) expression of NO-cGMP signaling (NO-cGMP 'producers'/'effectors'/'removers'). The results showed that oral administration of α1-ADR blocker before stress increased cGMP and diminished stress-reduced cAMP production in Leydig cells. In the same cells, stress-induced effects on cAMP/cGMP signaling pathways elements were changed. Sustained in vivo α1-ADR blockade completely abolished stress-increased transcription of most abundantly expressed phosphodiesterase that remove cAMP (Pde4b) and potentiated stress-increased expression of PRKA, the main stimulator of Leydig cell steroidogenesis. In the same Leydig cells, stress-decreased NOS3 expression was abolished, while stress-increased GUCY1 (cGMP 'producer') and PRKG1 (cGMP 'effector') were potentiated. It is possible that all molecules mentioned could contribute, at least in part, in recovery of Leydig cell testosterone production. Presented data provide new role of α1-ADRs in stress-triggered disturbance of cAMP/cGMP signaling, and new molecular insights into the relationship between stress and mammalian reproduction. Regardless of whether the effects of α1-blocker + stress are direct or indirect, the results are important in terms of human reproductive health and the wide use of α1-ADR antagonists, alone or in combination, to treat post-traumatic stress disorders, hypertension, benign prostatic hyperplasia symptoms and potential drugs for prostate cancer prevention/treatment.

The opposing roles of nitric oxide and cGMP in the age-associated decline in rat testicular steroidogenesis.

The molecular mechanism of the aging-associated dysfunction of Leydig cells (LCs) is complex and poorly understood. In this study, we analyzed the contribution of nitric oxide (NO) and cGMP signaling to the age-dependent decline in LC function. Significant (>50%) decreases in serum, intratesticular, and LC androgens in aging rats (15-24 months) were accompanied by a proportional increase in NO production, an up-regulation of cGMP levels, and the expression of soluble guanylyl cyclase-1B and protein kinase G1 in LCs. In contrast, LC cAMP levels decreased with age, most likely reflecting the up-regulation of cAMP-specific phosphodiesterase expression. Moreover, the expression of genes encoding enzymes responsible for cholesterol transport and its conversion to T were reduced. Exposing LCs from aged animals to NO further increased cGMP levels and decreased cAMP and androgen production, whereas the addition of cell-permeable 8-bromoguanosine-cGMP alone had the opposite effect. In vivo inhibition of cGMP-specific phosphodiesterase-5 for 3 and 6 months in aged rats led to a partial restoration of androgens, NO, and cyclic nucleotide levels, as well as the expression of steroidogenic and NO/cGMP signaling genes. These results indicate that a progressive increase in NO production contributes to the age-dependent decrease in steroidogenesis in a cGMP-independent manner, whereas the sustained elevation in cGMP levels significantly slows the decline in LC function.