adenylate cyclase activator
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2020 ◽  
Vol 32 (11) ◽  
pp. 1012
Author(s):  
Elizabeth S. Metcalf ◽  
Keith R. Masterson ◽  
David Battaglia ◽  
Jeremy G. Thompson ◽  
Robert Foss ◽  
...  

Optimising the developmental potential of immature equine oocytes and invitro-produced (IVP) embryos was explored through modifications of established media and holding temperature. In Experiment 1, delaying spontaneous resumption of meiosis through the process of simulated physiological oocyte maturation with the addition of the adenylate cyclase activator forskolin (50µM) and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (100µM) to overnight holding medium before maturation improved blastocyst production (P<0.05). In Experiment 2, the blastocyst production rate was increased significantly when cumulin (100ng mL−1) was added to the overnight holding or culture media (P<0.05). In Experiment 3, immature oocytes held overnight at 16°C before maturation had improved developmental competence than those held at 20°C and 5°C (P<0.05). There was no difference between maturation rates, but blastocyst formation per cleaved oocyte was significantly greater in oocytes held overnight at 16°C than at 20°C or 5°C. Furthermore, blastocyst formation per recovered oocyte and per fertilised oocyte was greater when oocytes were held before maturation at 16°C than at 5°C (P<0.05). In Experiment 4, the addition of sodium ascorbate (AC; 50µg mL−1) to the maturation and/or culture media of oocytes and IVP embryos did not improve blastocyst production, but did appear to lower cleavage rates compared with oocytes and embryos cultured without AC.


2019 ◽  
Vol 317 (1) ◽  
pp. C131-C142 ◽  
Author(s):  
Zhihui Fong ◽  
Caoimhín S. Griffin ◽  
Mark A. Hollywood ◽  
Keith D. Thornbury ◽  
Gerard P. Sergeant

β3-Adrenoceptor (β3-AR) agonists are used to treat overactive bladder syndrome; however, their mechanism of action has not been determined. The aims of this study were to compare the effects of β3-AR agonists on cholinergic versus purinergic receptor-mediated contractions of the detrusor and to examine the mechanisms underlying inhibition of the purinergic responses by β3-AR agonists. Isometric tension recordings were made from strips of murine detrusor and whole cell current recordings were made from freshly isolated detrusor myocytes using the patch-clamp technique. Transcriptional expression of exchange protein directly activated by cAMP (EPAC) subtypes in detrusor strips was assessed using RT-PCR and real-time quantitative PCR. The β3-AR agonists BRL37344 and CL316243 (100 nM) inhibited cholinergic nerve-mediated contractions of the detrusor by 19 and 23%, respectively, but did not reduce contractions induced by the cholinergic agonist carbachol (300 nM). In contrast, BRL37344 and CL316243 inhibited purinergic nerve-mediated responses by 55 and 56%, respectively, and decreased the amplitude of contractions induced by the P2X receptor agonist α,β-methylene ATP by 40 and 45%, respectively. The adenylate cyclase activator forskolin inhibited purinergic responses, and these effects were mimicked by a combination of the PKA activator N6-monobutyryl-cAMP and the EPAC activator 8-pCPT-2′- O-methyl-cAMP-AM (007-AM). Application of ATP (1 μM) evoked reproducible P2X currents in isolated detrusor myocytes voltage-clamped at −60 mV. These responses were reduced in amplitude in the presence of BRL37344 and also by 007-AM. This study demonstrates that β3-AR agonists reduce postjunctional purinergic responses in the detrusor via a pathway involving activation of the cAMP effector EPAC.


2010 ◽  
Vol 22 (9) ◽  
pp. 94
Author(s):  
M. Bertoldo ◽  
T. Sellens ◽  
C. G. Grupen

Asynchronous nuclear and cytoplasmic maturation is thought to contribute to poor embryo production in vitro. Nuclear arrest is mediated by cAMP and can be maintained within the oocyte using non-specific phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine ; IBMX) and the adenylate cyclase activator forskolin (FSK) (1). The aim of this study was to investigate the effect of IBMX and FSK supplementation on porcine oocyte nuclear maturation during COC recovery and IVM using a defined culture system. In all experiments, cAMP modulators were added to Hepes-buffered media held in collection tubes. COCs recovered from 3–5 mm diameter follicles of prepubertal ovaries were cultured in basic maturation media in the absence of FSH. Nuclear maturation was assessed using orcein dye. In Experiment 1, IVM media was supplemented with 0, 50 or 500 µM IBMX. In Experiment 2, IVM media was supplemented with 0, 5, 10, 50 and 100µM FSK. In Experiment 3, IVM medium was supplemented with combinations of IBMX and FSK to give the treatments; control, 50IBMX/50FSK, 50IBMX/100FSK, 500IBMX/50FSK and 500IBMX/100FSK. Nuclear maturation was assessed at 0, 2, 4 and 18 h after the onset of IVM. At 18 h of culture, there were no differences in the proportion of oocytes supplemented with 0, 50 or 500 µM IBMX reaching MII. Incubation with 10, 50 or 100 µM FSK resulted in 8-16% of oocytes at MII at 18 h compared to the other groups (25–29%; P < 0.001). The combinations of IBMX and FSK resulted in greater proportions (86–98%) of oocytes remaining at the GV stage at 18 h compared to the control (16%; P < 0.001). There were no differences in the proportion of oocytes remaining at the GV stage at the earlier time points (P > 0.05). The results demonstrate that these cAMP modulators, in combination, are highly effective in maintaining porcine oocyte meiotic arrest in vitro for an extended period. (1) Albuz FK et al., Proceedings of the 25th Annual Meeting of ESHRE, Amsterdam, The Netherlands, 2009.


2008 ◽  
Vol 294 (6) ◽  
pp. G1376-G1383 ◽  
Author(s):  
Jianhua Ren ◽  
Xiaoping Zhou ◽  
James J. Galligan

5-HT4 receptor agonists facilitate synaptic transmission in the enteric nervous system, and these drugs are used to treat constipation. In the present study, we investigated the effects of the 5-HT4 receptor agonist, renzapride, on rundown and recovery of fast excitatory postsynaptic potentials (fEPSPs) during and after trains of stimulation and on transmitter release from individual myenteric neuronal varicosities. Intracellular electrophysiological methods were used to record fEPSPs from neurons in longitudinal muscle myenteric plexus preparations of guinea pig ileum in vitro. During trains of supramaximal electrical stimulation (10 Hz, 2 s), fEPSP amplitude declined (time constant = 0.6 ± 0.1 s) from 17 ± 2 mV to 0.7 ± 0.3 mV. Renzapride (0.1 μM) did not change the time constant for fEPSP rundown, but it decreased the time constant for recovery of fEPSP amplitude after the stimulus train from 7 ± 2 s to 1.6 ± 0.2 s ( P < 0.05). 5-HT (0.1 μM) also increased fEPSPs and facilitated recovery from rundown. The adenylate cyclase activator, forskolin (1 μM), mimicked the actions of renzapride and 5-HT, whereas H-89, a protein kinase A (PKA) inhibitor, blocked the effects of renzapride. We used nicotinic acetylcholine receptor containing outside-out patches obtained from myenteric neurons maintained in primary culture to detect acetylcholine release from single varicosities. Renzapride (0.1 μM) increased release probability twofold. We conclude that 5-HT4 receptors activate the adenylyl cyclase-PKA pathway to increase acetylcholine release from single varicosities and to accelerate recovery from synaptic rundown. These responses may contribute to the prokinetic actions of 5-HT4 receptor agonists.


1998 ◽  
Vol 275 (2) ◽  
pp. E213-E221
Author(s):  
Wenhan Chang ◽  
Tsui-Hua Chen ◽  
Stacy Pratt ◽  
Dolores Shoback

Parathyroid cells express Ca2+-sensing receptors that couple changes in the extracellular Ca2+ concentration ([Ca2+]o) to increases in the intracellular free Ca2+ concentration ([Ca2+]i) and to the suppression of parathyroid hormone secretion. Using whole cell patch clamping, we previously identified voltage-independent Ca2+-conducting currents in bovine parathyroid cells that increased with rising [Ca2+]oand were blocked by Cd2+ and nifedipine. Because cAMP-dependent phosphorylation regulates dihydropyridine-sensitive Ca2+channels in other systems, we tested whether cAMP modulates these currents. At 0.7 mM Ca2+, nonselective Ca2+-conducting currents were suppressed by 30–50% when the recording pipette was perfused with cAMP. High-[Ca2+]o-induced increases in membrane currents were also abrogated. The effects of cAMP were reversible and dose dependent (3 × 10−9 to 3 × 10−3 M) and required ATP in the pipette solution. Perfusion of the cell interior with the catalytic subunit of protein kinase A mimicked the effects of cAMP, as did perfusion of the bath with the adenylate cyclase activator forskolin. These findings support the idea that cAMP-dependent phosphorylation suppresses high-[Ca2+]o-induced cation currents and may play a role in regulating ion fluxes in parathyroid cells.


1997 ◽  
Vol 273 (4) ◽  
pp. C1349-C1353 ◽  
Author(s):  
Gabriela Picotto ◽  
Virginia Massheimer ◽  
Ricardo Boland

Direct effects of parathyroid hormone (PTH) on calcium uptake by isolated rat duodenal cell preparations enriched in enterocytes were investigated. PTH significantly stimulated enterocyte45Ca2+influx in a time-dependent (1–10 min) manner and at all doses tested (2 × 10−13 to 10−7 M). The Ca2+ channel antagonists verapamil (10 μM) and nitrendipine (1 μM) completely blocked the stimulation of Ca2+ influx by the hormone (10−8 M). PTH markedly increased cAMP levels in rat duodenal cells (88, 167, and 67%, after 1, 2, and 3 min, respectively). In agreement with these observations, forskolin (adenylate cyclase activator), dibutyryl adenosine 3′,5′-cyclic monophosphate (DBcAMP), and Sp-cAMPS (cAMP analogs) mimicked, whereas Rp-cAMPS (cAMP antagonist) suppressed PTH and DBcAMP activation of enterocyte calcium uptake. Furthermore, the effects of DBcAMP were abolished by nitrendipine. These results show direct rapid effects of PTH on duodenal cells’ Ca2+ influx, which involve the activation of a dihydropyridine-sensitive Ca2+ influx pathway and the cAMP second messenger system.


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