The hypothalamic peptide, pituitary adenylate cyclase-activating polypeptide (PACAP), can efficiently increase cAMP levels in pituitary cells and release a number of pituitary hormones, suggesting an important physiological role for this peptide in pituitary function. Exposure of GH3 rat pituitary cells to PACAP results in increases in cellular cAMP levels, PRL promoter activity, and PRL messenger RNA levels. We have employed this system to further characterize PACAP regulation of PRL gene expression. RT-PCR analysis showed that GH3 cells express transcripts for two PACAP receptors, PACAP-R-hop1 and VIP2. As the former can couple PACAP to increases in both cAMP and inositol phosphates, we investigated whether either pathway mediates PACAP action on the PRL promoter. Our observations that TRH, but not PACAP, increases the intracellular Ca2+ concentration in GH3 cell cultures and that the optimal concentrations of TRH and PACAP have additive effects on transient expression of a PRL-CAT construct imply that the inositol trisphosphate-Ca2+ pathway is not significantly involved in PACAP action on the PRL promoter. Four kinase inhibitors exhibited similar profiles of inhibition of the activity on PRL-chloramphenicol acetyltransferase (PRL-CAT) of either the adenylyl cyclase activator forskolin (FSK) or PACAP, suggesting a transcriptional role for protein kinase A (PKA). The observations that coexpression of the dominant PKA inhibitor RAB completely blocked either FSK or PACAP action on PRL-CAT and that these actions of FSK and PACAP were completely nonadditive imply that the cAMP-PKA pathway plays a dominant role in PACAP regulation of PRL gene expression. Coexpression of low levels of KCREB, a cAMP response element (CRE)-binding protein (CREB) dominant inhibitor, partially blocked regulation of PRL-CAT activity by PACAP, but not TRH, implying that PACAP action is mediated at least in part by a CREB family member that can dimerize with CREB. The PRL promoter contains an asymmetric sequence at positions -99/-92 resembling a canonical CRE and termed here the CRE-like element (CLE). Mutation of either the left or right 4 bp of the CLE yielded a strong decrease in the response to either FSK or PACAP, but not to TRH. These data imply that PACAP and TRH employ independent pathways to regulate the PRL promoter, and that PACAP action is exerted virtually entirely via a cAMP/PKA-mediated pathway that is strongly dependent upon an intact CLE sequence and at least partially dependent upon the activity of a CREB-related protein.
Measuring instruments are intended to be intelligent, precise, multi-functional and developing multidirectionally, scientific, and reasonable; the reliable evaluation of measurement uncertainty of precision instruments is also becoming more and more difficult, and the evaluation of the Coordinate Measuring Machines (CMM) measurement uncertainty is among the typical problems. Based on Geometric Product Specification (GPS), this paper has systematically studied the CMM uncertainty for evaluating the size and geometrical errors oriented toward measurement tasks, and thus has realized the rapid and reliable evaluation of the CMM uncertainty for task-oriented measurement. For overestimation of the CMM uncertainty for task-oriented measurements in the initial evaluation, a systematic optimization solution has been proposed. Finally, the feasibility and validity of the evaluation model and the optimization method have been verified by three different types of measurement examples of diameter, flatness and perpendicularity. It is typical and representative to systematically solve the problem of the CMM uncertainty for evaluating the measurement tasks targeted at dimensions and geometric errors, and the research contents can be effectively applied to solve the uncertainty evaluation problems of other precision instruments, which are of great practical significance not only for promoting the combination of modern uncertainty theory and practical applications but also for improving the application values of precision measurement instruments.
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