The steroidogenic activities and receptor-binding properties of angiotensin II and des-Asp1-angiotensin II were analyzed and compared with the metabolism of each peptide during incubation with isolated rat and canine adrenal glomerulosa cells. In isolated rat glomerulosa cells, equimolar concentrations of each peptide stimulated aldosterone production similarly over the first 20 min of incubation. However, at later times, the des-Asp1 heptapeptide was consistently less active, with one third the potency of the octapeptide at 40 min, and only one tenth that of the octapeptide after 120 min. In dog glomerulosa cells, the des-Asp1 heptapeptide was less rapidly inactivated and retained one third the potency of the octapeptide after 120 min of incubation. In each species, the maximum aldosterone response to the two peptides was similar at all time intervals studied up to 120 min. Also, no additivity between maximal concentrations of angiotensin II and des-Asp1-angiotensin II was observed. Assay of angiotensin II receptors with radioiodinated and tritiated peptides in rat glomerulosa cells and adrenal particles showed identical receptor concentration for each peptide, with somewhat lower affinity for the heptapeptide (Ka = 0.7 × 109 M-1 for the heptapeptide vs. 2.7 × 109 M-1 for angiotensin II). The metabolism of angiotensin in isolated glomerulosa cells was analyzed by thin layer chromatography and assay of peptides in the incubation medium. Extensive degradation of angiotensin II during incubation with rat cells was detected, with relatively little conversion to the 2–8 and 3–8 peptides. RIA of medium peptides showed decreases to 50% and 30% for angiotensin II and des-Asp1-angiotensin II at 40 min, and to 26% and 10% at 120 min, respectively. Similar results were derived with radioreceptor assay and bioassay by stimulation of aldosterone production in fresh glomerulosa cells. In contrast, metabolism of angiotensin during incubation with dog adrenal glomerulosa cells was less rapid, and both peptides were degraded at the same rate. Analysis of labeled peptides eluted from rat glomerulosa cells after in vivo or in vitro binding of [125I]iodoangiotensin II showed that more than 90% of the bound radioactivity was composed of the octapeptide. These studies have revealed extensive metabolism of angiotensin peptides by isolated rat glomerulosa cells, with more rapid degradation of des-Asp1-angiotensin II than of the native octapeptide. Such peptide metabolism contributes to the apparent discrepancy in biological potencies of angiotensin II and des-Asp1-angiotensin II on aldosterone production in isolated rat glomerulosa cells. It is also evident that angiotensin II acts upon rat and dog glomerulosa cells to evoke aldosterone production without prior conversion to the heptapeptide.
Testing the hypothesis that a sleep-light intervention, which phase-advances melatonin rhythms, will improve perimenopausal-postmenopausal (P-M; by follicle-stimulating hormone) depression.
In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF1 receptor (CRF1R) activation in transfected COS-7 cells. We found that Pertussis Toxin (PTX) treatment or sequestering Gβγ reduced CRF1R-mediated activation of ERK1/2, suggesting the involvement of a Gi-linked cascade. Neither Gs/PKA nor Gq/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr1068, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRF1R-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a Gβγ-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr845) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRF1R, Src, and EGFR facilitates EGFR transactivation and CRF1R-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on Gi/βγ subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRF1R ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRF1R and the phosphorylation-deficient CRF1R-Δ386 mutant, which has impaired agonist-dependent β-arrestin-2 recruitment; however, this situation may have resulted from the low β-arrestin expression in the COS-7 cells. When β-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRF1R/EGFR interaction, the Gi/βγ subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRF1R signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRF1R is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.
