The mechanisms underlying AVP‐induced increase in [Ca 2+ ] i and glucagon release in clonal α‐cells In‐R1‐G9 were investigated. AVP increased [Ca 2+ ] i and glucagon release in a concentration‐dependent manner. After the administration of AVP, glucagon was released within 30 s, quickly reached the maximum within 2 min, and maintained a steady‐state concentration for at least 15 min. In Ca 2+ ‐containing medium, AVP increased [Ca 2+ ] i in a biphasic pattern; a peak followed by a sustained plateau. In Ca 2+ ‐free medium, the Ca 2+ response to AVP became monophasic with lower amplitude and no plateau. Both the basal and AVP‐induced glucagon releases were lower in the absence than in the presence of extracellular Ca 2+ . When [Ca 2+ ] i was stringently deprived by BAPTA, a Ca 2+ chelator, AVP still significantly increased glucagon release. Pretreatment with thapsigargin, a microsomal Ca 2+ ATPase inhibitor, abolished both the Ca 2+ peak and sustained plateau. AVP increased intracellular concentration of IP 3 . U‐73122 (8 μ M ), a phospholipase C inhibitor, abolished AVP‐induced increases in [Ca 2+ ] i , but only reduced AVP‐induced glucagon release by 39%. Pretreatment with nimodipine, an L‐type Ca 2+ channel blocker failed to alter AVP‐induced glucagon release or increase in [Ca 2+ ] i . The results suggest that AVP causes glucagon release through both Ca 2+ ‐dependent and ‐independent pathways. For the Ca 2+ ‐dependent pathway, the G q protein activates phospholipase C, which catalyzes the formation of IP 3 . IP 3 induces Ca 2+ release from the endoplasmic reticulum, which, in turn, triggers Ca 2+ influx. Both Ca 2+ release and Ca 2+ influx may contribute to AVP‐induced glucagon release. British Journal of Pharmacology (2000) 129 , 257–264; doi: 10.1038/sj.bjp.0703037
We tested the hypothesis that the formamidine pesticide amitraz and its metabolites inhibit insulin release from a rat beta-cell line (RINm5F) by decreasing intracellular cyclic AMP levels and examined whether GTP-binding proteins were involved in the process. Amitraz and its active metabolite BTS27271 (0.1-10 microM) inhibited insulin release that was induced by 100 microM of IBMX in a dose-dependent manner. Other metabolites of amitraz tested failed to inhibit insulin release. A potent and specific alpha-2 adrenoceptor agonist, medetomidine (0.1 microM), abolished the IBMX-induced insulin release. Amitraz, BTS27271 and medetomidine also decreased intracellular cyclic AMP concentrations that were elevated by IBMX administration. Moreover, all three drugs inhibited the insulin release stimulated by forskolin (1 microM), an adenylyl cyclase activator. Yohimbine (0.01, 0.1 and 1 microM), an alpha-2 adrenoceptor antagonist, prevented the inhibitory effect of amitraz and BTS27271 on insulin release, whereas prazosin (1 microM), an alpha-1 adrenoceptor antagonist, did not. Yohimbine, but not prazosin, also prevented the effect of amitraz, BTS27271 and medetomidine on IBMX-stimulated accumulation of cyclic AMP. Pretreatment of cells with PTX (0.1 micrograms/ml) for 16 h, antagonized the effects of amitraz and BTS27271 on IBMX-induced increase in insulin release. Thus, one mechanism by which amitraz and its metabolite BTS27271 decrease insulin release is by inhibiting adenylyl cyclase, an action mediated through a PTX-sensitive G-protein.
The present study investigated the mechanism by which arginine vasopressin (AVP) increases insulin secretion in rat insulinoma (RINm5F) cells by using a specific phospholipase C (PLC) inhibitor, 1-[6-[[17 beta-3-methoxyestra-1,3,5(10)-trien-17- yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), and a phospholipase A2 (PLA2) inhibitor, N-(p-amylcinnamoyl)anthranilic acid (ACA). AVP (0.1-100 nM) increased insulin secretion and cytosolic free Ca++ concentration ([Ca++]i) dose-dependently. AVP-induced increases in the intracellular concentration of inositol 1,4,5-trisphosphate (IP3) and [Ca++]i were dose-dependently inhibited by U-73122 (2-8 microM). At 8 microM, U-73122 abolished AVP's effect on IP3 and [Ca++]i, but AVP-induced increases in insulin secretion were only reduced by 35%. In contrast, 8 microM U-73122 did not reduce the ionomycin (a Ca++ ionophore, 100 nM)-induced increase in [Ca++]i. The discrepancy between the results of [Ca++]i and insulin secretion in U-73122 experiments is indicative of the multiple signal transduction pathways associated with the activation of AVP receptors, specifically the Ca(++)-independent pathway. The phospholipase A2 inhibitor ACA (100 microM) did not antagonize AVP (10 nM)-induced increases in insulin secretion. These results suggested: 1) U-73122 blocks PLC activities but fails to block other signal transduction pathways that trigger insulin secretion in these cells and 2) AVP increases insulin release from RINm5F cells through both the PLC-mediated Ca(++)-dependent and Ca(++)-independent pathways.
Background: It is known that quantification of bovine acute‐phase proteins could routinely provide early diagnostic and prognostic information for monitoring herd health status. Objective: The aim of this study was to test the hypothesis that haptoglobin (Hp) and serum amyloid A (SAA) have the potential for indicating uterine infection, mainly due to subclinical endometritis, which can seriously influence the reproductive performance in dairy cows. Methods: A total of 264 serum samples were collected from 24 Holstein dairy cows at defined intervals from 1 week prepartum to 6 months postpartum. Clinically healthy animals (Group 1, n =6) were compared with 24 animals having acute puerperal metritis (Group 2, n =18) and with concentrations obtained from healthy heifers (baseline values, n =10). The concentrations of serum Hp and SAA were measured using a hemoglobin‐binding assay and Western blot analysis, respectively. Reproductive performance was assessed as the number of days open and the conception rate at the end of the study. Results: Upper cut‐off values for Hp and SAA concentrations in heifers were 130.9 μg/L and 51.9 μg/mL, respectively. Hp concentrations in Group 2 were significantly higher than those in Group 1 at all time points ( P <.001). Additionally, among the successfully pregnant animals, the number of days open was significantly higher in 6 cows with Hp >130.9 μg/mL (median=316 days) than in 11 cows with Hp ≤130.9 μg/mL (median=120 days) ( P =.0024). Conclusions: Serum Hp and SAA concentrations can be used to recognize uterine infection in postpartum cows. The association between elevated Hp values and number of days open suggests Hp may also be a useful indicator of poor prognosis for reproductive performance.
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