Inhibitory Effect Of Forskolin Research Articles

Cyclic adenosine monophosphate-dependent activation of transient receptor potential vanilloid 4 (TRPV4) channels in osteoblast-like MG-63 cells

Parathyroid hormone (PTH) directly interacts with bone remodeling osteoblasts and osteocytes expressing the G-protein coupled receptor PTH receptor 1 (PTH1R), and its osteoanabolic effects mostly involve the cAMP/PKA signaling cascade. Considering that PTH-dependent calcium entry in rat enterocytes is reproduced by the adenylate cyclase agonist forskolin or by cAMP analogues, possible involvement of calcium as a second messenger in PTH-dependent cAMP signaling was investigated in MG-63 cells. First, Ca2+ influx was confirmed in Fluo3-loaded MG-63 cells treated with a cell-permeable cAMP analog. Second, PTH (1–34) and forskolin promoted calcium influxes that were completely abrogated by the PKA inhibitor H-89. Ca2+ entry was not reproduced when PTH (1–34) was combined with the PKC-activating competitor PTH (3–34). Vanilloid transient potential (TRPV) channel inhibitor Ruthenium Red, but not a voltage-dependent calcium channel (VDCC) inhibitor nifedipine, efficiently stunted Ca2+ entry, and comparable abrogation was reproduced in cells treated with TRPV4-selective inhibitor RN-1734 or transfected with TRPV4-specific siRNA. Interestingly, PTH-driven Ca2+ through TRPV4 significantly inhibited MG63 cell migration through a mechanism requiring extracellular Ca2+. In contrast, the inhibitory effects of forskolin on migration were refractory to TRPV4 silencing or to RN-1734. Altogether, our results indicate that single treatment with PTH (1–34) promotes extracellular calcium entry through TRPV4 channels in MG-63 cells through a cAMP/PKA-dependent mechanism, and that this influx affects cell migration.

Role of Myosins II and VI in Mediating Hormonal Regulation of NHE3 Activity in the Rat Renal Proximal Tubule

Rapid regulation of NHE3 activity may occur through redistribution of the transporter between the body and the base of the proximal tubule microvilli (PT MV). Myosins are motor proteins that move along actin filaments. The present study tested the hypothesis that myosins II (myo II), which moves cargo away from cell center, and VI (myo VI), which moves cargo toward cell center, may mediate NHE3 redistribution within the MV in response to stimuli. Renal PT NHE3 activity was evaluated in Wistar rats by net bicarbonate reabsorption (JHCO3−) in the presence or absence of 10−10 M angiotensin II (Ang II), 10−4 M forskolin (FSK), a cAMP‐elevating agent, 10−5 M blebbistatin (a specific myo II inhibitor) or 4×10−6 M 2,4,6‐triiodophenol (TIP) (a myo VI inhibitor). As expected, Ang II increased [4.36 ± 0.18 nmol/cm2.s (N = 5)] whereas FSK reduced [1.26 ± 0.10 nmol/cm2.s (N = 5)] JHCO3− compared to CTRL [2.74 ± 0.13 nmol/cm2.s (N = 4)]. Simultaneous tubular perfusion of Ang II with blebbistatin prevent Ang II‐induced NHE3 stimulation [2.92 ± 0.14 nmol/cm2.s (N = 5), P < 0.001]. Simultaneous tubular perfusion of FSK with TIP reduced the inhibition with FSK alone [1.78 ± 0.15 (N = 4) vs. 1.26 ± 0.10 nmol/cm2.s (N = 5), P < 0.05] on JHCO3−. The localization of NHE3 and myosins IIB and VI was assessed by confocal immunofluorescence in Wistar rats kidney after a 30 min pressor dose of Ang II (50 ng/kg/min). NHE3 and myo VI shift from base to body of MV after Ang II; myo IIB remains mostly enriched at the MV base. To assess NHE3 activity in OKP cells, experimental pH recovery points were fitted to simple exponential decay curves (y = A·e(−kt) + y0) in order to predict the rate constant (k) of each recovery curve. FSK reduced NHE3 activity [0.025 ± 0.003 s−1 (N = 4)] compared to CTRL [0.048 ± 0.007 s−1 (N = 5)]. siRNA for myo VI efficiently reduced its expression by 64 %; the inhibitory effect of FSK on NHE3 activity was completely abolished by siRNA myo VI [0.043 ± 0.005 s−1 (N = 4)]. These results provide further evidence for myosin II and VI regulation of NHE3 redistribution in the rat kidney proximal tubule.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential regulation of kit ligand A (kitlga) expression in the zebrafish ovarian follicle cells – Evidence for the existence of a cyclic adenosine 3′, 5′ monophosphate-mediated binary regulatory system during folliculogenesis

Kit ligand (Kitl) is an important paracrine factor involved in the activation of primordial follicles from the quiescent pool and in the maintenance of meiotic arrest before germinal vesicle breakdown (GVBD). It has been reported that follicle-stimulating hormone (FSH) stimulates but luteinizing hormone (LH) suppresses the expression of Kitl in the granulosa cells in mammals. Considering that both gonadotropins signal in the follicle cells mainly by activating cyclic adenosine 3′, 5′-monophosphate (cAMP) pathway, we are intrigued by how cAMP differentially regulates Kitl expression. In the present study, we demonstrated that both human chorionic gonadotropin (hCG) and pituitary adenylate cyclase activating polypeptide (PACAP) inhibited insulin-like growth factor I (IGF-I)-induced Akt phosphorylation and kitlga expression in the zebrafish follicle cells. Further experiments showed that cAMP was involved in regulating the expression of kitlga. However, two cAMP-activated effectors, protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), had converse effects. PKA promoted whereas Epac inhibited the expression of kitlga, as demonstrated by the respective activators. Interestingly, cAMP also appeared to exert differential effects on kitlga expression at different stages of follicle development during folliculogenesis, significantly stimulating kitlga expression at the early growth stage but suppressing it at the full-grown stage before final oocyte maturation, implying a potential mechanism for differential effects of the same pathway at different stages. The inhibitory effect of forskolin (activator of adenylate cyclase) and H89 (inhibitor of PKA) on IGF-I-induced expression of kitlga suggested cross-talk between the cAMP and IGF-I-activated PI3K-Akt pathways. This study, together with our previous findings on IGF-I regulation of kitlga expression, provides important clues to the underlying mechanism that regulates Kit ligand expression during folliculogenesis in the ovary.

Targeted activation of PKA and Epac promotes glioblastoma regression in vitro

Ras-p44/42 mitogen-activated protein kinase (MAPK) and Akt signaling are the key pathways involved in the promotion of glioblastoma formation. Notably, phosphodiesterase 4 (PDE4) is widely expressed in brain tumors and promotes their growth. PDE4 inhibitors have been reported to suppress glioblastoma growth in vitro and in vivo. The mechanisms underlying these actions, however, have yet to be elucidated. The aim of this study was to investigate whether intracellular cyclic adenosine monophosphate (cAMP) was able to suppress the Ras-p44/42 MAPK signaling pathway via protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac) in U87MG human malignant glioma cells. Forskolin, an activator of adenylate cyclase, inhibited cell growth and the phosphorylation of p44/42 MAPK in U87MG cells, whereas the non-hydrolyzable cAMP analog 8-bromoadenosine 3',5'-cAMP (8-Br-cAMP) considerably suppressed cell growth and phosphorylation of p44/42 MAPK. The inhibitory effects of forskolin were partially prevented by the PKA inhibitor H89. The Epac-selective agonist 8-(4-chlorophenylthio)-2'-O-methyladenosine cAMP (8-CPT-cAMP) inhibited phosphorylation of p44/42 MAPK. These findings suggest that PKA and Epac are involved in the effect of intracellular cAMP on the Ras-p44/42 MAPK signaling pathway.

Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway Inhibits Endothelin Type A Receptor-Operated Ca2+ Entry Mediated via Transient Receptor Potential Canonical 6 Channels

Receptor-operated Ca²⁺ entry (ROCE) via transient receptor potential canonical channel 6 (TRPC6) is important machinery for an increase in intracellular Ca²⁺ concentration triggered by the activation of G(q) protein-coupled receptors. TRPC6 is phosphorylated by various protein kinases including protein kinase A (PKA). However, the regulation of TRPC6 activity by PKA is still controversial. The purpose of this study was to elucidate the role of adenylate cyclase/cAMP/PKA signaling pathway in the regulation of G(q) protein-coupled endothelin type A receptor (ET(A)R)-mediated ROCE via TRPC6. For this purpose, human embryonic kidney 293 (HEK293) cells stably coexpressing human ET(A)R and TRPC6 (wild type) or its mutants possessing a single point mutation of putative phosphorylation sites for PKA were used to analyze ROCE and amino acids responsible for PKA-mediated phosphorylation of TRPC6. Ca²⁺ measurements with thapsigargin-induced Ca²⁺-depletion/Ca²⁺-restoration protocol to estimate ROCE showed that the stimulation of ET(A)R induced marked ROCE in HEK293 cells expressing TRPC6 compared with control cells. The ROCE was inhibited by forskolin and papaverine to activate the cAMP/PKA pathway, whereas it was potentiated by Rp-8-bromoadenosine-cAMP sodium salt, a PKA inhibitor. The inhibitory effects of forskolin and papaverine were partially cancelled by replacing Ser28 (TRPC6(S28A)) but not Thr69 (TRPC6(T69A)) of TRPC6 with alanine. In vitro kinase assay with Phos-tag biotin to determine the phosphorylation level of TRPC6 revealed that wild-type and mutant (TRPC6(S28A) and TRPC6(T69A)) TRPC6 proteins were phosphorylated by PKA, but the phosphorylation level of these mutants was lower (approximately 50%) than that of wild type. These results suggest that TRPC6 is negatively regulated by the PKA-mediated phosphorylation of Ser28 but not Thr69.

Adiponectin inhibits palmitate-induced apoptosis through suppression of reactive oxygen species in endothelial cells: involvement of cAMP/protein kinase A and AMP-activated protein kinase

The present study examined whether adiponectin can inhibit palmitate-induced apoptosis, and also the associated mechanisms and signal transduction pathways in human umbilical vein endothelial cells. Cells treated with 500 μM palmitate for 48 h increased reactive oxygen species (ROS) generation and induced apoptosis. Treatment with antioxidant N-acetyl-L-cysteine (1 mM) and globular adiponectin (5 μg/ml) inhibited palmitate-induced ROS generation and apoptosis. The AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR; 1 mM), and cAMP activators forskolin (10 μM) and cholera toxin (200 ng/ml) also displayed the same effects. The inhibitory effects of adiponectin on ROS generation and apoptosis were reversed by the AMPK inhibitor compound C (40 μM), cAMP inhibitor SQ22536 (50 μM), and protein kinase A (PKA) inhibitor H-89 (10 μM). The inhibitory effect of forskolin on palmitate-induced apoptosis was reversed by compound C, whereas the inhibitory effect of AICAR was not reversed by SQ22536 and H-89. AICAR and forskolin could not inhibit palmitate-induced apoptosis in cells treated with dominant-negative AMPK. Forskolin increased phosphorylated AMPK at both Thr-172 and Ser-485/491. These results suggest that adiponectin inhibits palmitate-induced apoptosis by suppression of ROS generation via both the cAMP/PKA and AMPK pathways. Interaction between cAMP/PKA and AMPK pathways may be involved.

Role of cAMP-dependent protein kinase in the regulation of platelet procoagulant activity

The membrane microparticle (MP) formation and phosphatidylserine (PS) exposure evoked by platelet activation provide catalytic surfaces for thrombin generation. Several reports have indicated the effects of cAMP-elevating agents on agonist-induced MP formation and PS exposure; however, the mechanism still remains unclear. Here we show that inhibition of basal cyclic AMP-dependent protein kinase (PKA) activity incurred platelet MP formation and PS exposure. Pretreatment of platelets with cAMP-elevating agent, forskolin, abolished thrombin plus collagen-induced MP formation and PS exposure, and obviously decreased calcium ionophore-evoked MP shedding. Moreover, the inhibitory effects of forskolin on agonists-induced MP formation and PS exposure were reversed by the PKA inhibitor H89. PKA inhibitor-induced MP formation was dose-dependently inhibited by calpain inhibitor MDL28170, and forskolin abrogated thrombin plus collagen-induced calpain activation. In conclusion, PKA plays key roles in the regulation of platelet MP formation and PS exposure. PKA-mediated MP shedding is dependent on calpain activation.

Differential Regulation of Endothelial Cell Permeability by cGMP via Phosphodiesterases 2 and 3

Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions, including edema and sepsis. Whereas cAMP has been shown to decrease endothelial permeability, the role of cGMP is controversial. Endothelial cells express cGMP-inhibited phosphodiesterase (PDE)3A and cGMP-stimulated PDE2A. Thus we hypothesized that the effect of cGMP on endothelial permeability is dependent on the concentration of cGMP present and on the relative expression levels of PDE2A and PDE3A. When cAMP synthesis was slightly elevated with a submaximal concentration of 7-deacetyl-7-(O-[N-methylpiperazino]-gamma-butyryl)-dihydrochloride-forskolin (MPB-forskolin), we found that low doses of either atrial natriuretic peptide (ANP) or NO donors potentiated the inhibitory effects of MPB-forskolin on thrombin-induced permeability. However, this inhibitory effect of forskolin was reversed at higher doses of ANP or NO. These data suggest that cGMP at lower concentrations inhibits PDE3A and thereby increases a local pool of cAMP, whereas higher concentrations cGMP activates PDE2A, reversing the effect. Inhibitors of PDE3A mimicked the effect of low-dose ANP on thrombin-induced permeability, and inhibition of PDE2A reversed the stimulation of permeability seen with higher doses of ANP. Finally, increasing PDE2A expression with tumor necrosis factor-alpha reversed the inhibition of permeability caused by low doses of ANP. As predicted, the effect of tumor necrosis factor-alpha on permeability was reversed by a PDE2A inhibitor. These findings suggest that the effect of increasing concentrations of cGMP on endothelial permeability is biphasic, which, in large part, is attributable to the relative amounts of PDE2A and PDE3A in endothelial cells.

NHE3 inhibition by cAMP and Ca2+ is abolished in PDZ‐domain protein PDZK1‐deficient murine enterocytes

The PDZ-binding protein PDZK1 (NHERF3/CAP70/PDZ-dc-1) in vitro binds to NHE3, but its role in the regulation of NHE3 activity in native enterocytes is unknown. This study was undertaken to understand the physiological role of PDZK1 in regulating NHE3 activity in native murine colonic enterocytes. NHE3 transport rates were assessed fluorometrically in BCECF-loaded colonic crypts in the NHE3-expressing cryptal openings by measuring acid-activated, Na+-dependent, Hoe 642-insensitive proton efflux rates. NHE3 mRNA expression levels and NHE3 total enterocyte and brush border membrane (BBM) protein abundance were determined by quantitative PCR and Western analysis and immunohistochemistry. In pdzk1-/- colonic surface cells, acid-activated NHE3 transport rates were strongly reduced, and the inhibitory effect of forskolin and ionomcyin was virtually abolished. Hyperosmolarity, on the other hand, still had an inhibitory effect. In addition, the NHE3-selective inhibitor S1611 inhibited acid-activated NHE3 activity in pdzk1-/- and +/+mice, suggesting that functional NHE3 is present in pdzk1-deficient colonocytes. NHE1 and NHE2 activity was not altered in pdzk1-/- colonic crypts. Immunohistochemistry revealed apical NHE3 staining in pdzk1-/- and +/+proximal colon, and Western blot analysis revealed no difference in NHE3 abundance in colonic enterocyte homogenate as well as brush border membrane. Lack of the PDZ-adaptor protein PDZK1 in murine proximal colonic enterocytes does not influence NHE3 abundance or targeting to the apical membrane, but abolishes NHE3 regulation by cAMPergic and Ca2+ -dependent pathways. It leaves NHE3 inhibition by hyperosmolarity intact, suggesting an important and selective role for PDZK1 in the agonist-mediated regulation of intestinal NHE3 activity.

Differential Regulation of Endothelial Cell Permeability by cGMP via Phosphodiesterases 2 and 3

Endothelial barrier dysfunction leading to increased permeability and vascular leakage is an underlying cause of several pathological conditions, including edema and sepsis. Whereas cAMP has been shown to decrease endothelial permeability, the role of cGMP is controversial. Endothelial cells express cGMP-inhibited phosphodiesterase (PDE)3A and cGMP-stimulated PDE2A. Thus we hypothesized that the effect of cGMP on endothelial permeability is dependent on the concentration of cGMP present and on the relative expression levels of PDE2A and PDE3A. When cAMP synthesis was slightly elevated with a submaximal concentration of 7-deacetyl-7-( O -[ N -methylpiperazino]-γ-butyryl)-dihydrochloride–forskolin (MPB–forskolin), we found that low doses of either atrial natriuretic peptide (ANP) or NO donors potentiated the inhibitory effects of MPB–forskolin on thrombin-induced permeability. However, this inhibitory effect of forskolin was reversed at higher doses of ANP or NO. These data suggest that cGMP at lower concentrations inhibits PDE3A and thereby increases a local pool of cAMP, whereas higher concentrations cGMP activates PDE2A, reversing the effect. Inhibitors of PDE3A mimicked the effect of low-dose ANP on thrombin-induced permeability, and inhibition of PDE2A reversed the stimulation of permeability seen with higher doses of ANP. Finally, increasing PDE2A expression with tumor necrosis factor-α reversed the inhibition of permeability caused by low doses of ANP. As predicted, the effect of tumor necrosis factor-α on permeability was reversed by a PDE2A inhibitor. These findings suggest that the effect of increasing concentrations of cGMP on endothelial permeability is biphasic, which, in large part, is attributable to the relative amounts of PDE2A and PDE3A in endothelial cells.

Modulation of voltage-dependent Ba2+ currents in the guinea-pig gastric antrum by cyclic nucleotide-dependent pathways.

We have investigated whether the activation of cAMP- and cGMP-dependent pathways modifies the properties of voltage-dependent Ba(2+) currents (I(Ba)) recorded from guinea-pig gastric myocytes using patch-clamp techniques. All experiments were carried on single smooth muscle cells, dispersed from the circular layer of the guinea-pig gastric antrum. Both dibutyryl cAMP (db-cAMP, 0.1-1 mM), a membrane-permeable ester of cAMP, and isoproterenol, a selective beta-stimulant, inhibited I(Ba) in a concentration-dependent manner. Forskolin, but not dideoxy-forskolin, an inactive isomer of forskolin, inhibited the peak amplitude of I(Ba). In the presence of either Rp-cAMP or the PKA (cAMP-dependent protein kinase) inhibitor peptide 5-24 (PKA-IP), neither forskolin nor db-cAMP inhibited I(Ba). After establishing a conventional whole-cell recording, the peak amplitude of I(Ba) gradually decreased when the catalytic subunit of PKA was included in the pipette. The further application of Rp-cAMP reversibly enhanced I(Ba). Sodium nitroprusside (0.1-1 mM) and 8-Br-cGMP (0.1-1 mM) also inhibited I(Ba) in a concentration-dependent manner. The inhibitory effects of forskolin or db-cAMP on I(Ba) were not significantly changed by pretreatment with a cGMP-dependent protein kinase (PKG) inhibitor. Similarly, the inhibitory actions of 8-Br-cGMP on I(Ba) were not modified by PKA-IP. The membrane-permeable cyclic nucleotides db-cAMP and 8-Br-cGMP caused little shift of the voltage dependence of the steady-state inactivation and reactivation curves. Neither of the membrane-permeable cyclic nucleotides db-cAMP or 8-Br-cGMP had additive inhibitory effects on I(Ba). These results indicate that two distinct cyclic nucleotide-dependent pathways are present in the guinea-pig gastric antrum, and that both inhibited I(Ba) in an independent manner.

Direct noncompetitive inhibition of 5-HT 3 receptor-mediated responses by forskolin and steroids

5-HT 3 receptors cloned from NCB-20 cells were expressed in Xenopus oocytes, and the effects of forskolin and steroids on the function of the receptors were investigated using the two-electrode voltage-clamp technique. Forskolin, 17-β-estradiol, and progesterone inhibited the currents activated by 1 μM 5-HT in a reversible and concentration-dependent manner, with IC 50 values of 12, 33, and 89 μM, respectively. The inhibitory effects of forskolin and 17-β-estradiol were independent of the membrane potential. Forskolin and 17-β-estradiol significantly reduced the maximal amplitude of the 5-HT concentration-response curve ( E max) without significantly affecting the EC 50, indicating that these compounds act as noncompetitive inhibitors of the 5-HT 3 receptor. The cAMP analogue, 8-Br-cAMP (0.2 mM), and the protein kinase A activator, Sp-cAMP (0.1 mM), did not affect the amplitude of 5-HT 3 receptor-mediated currents. The membrane-permeable protein kinase A inhibitor Rp-cAMP (0.1 mM) and the estrogen-receptor antagonist tamoxifen (1 μM) did not affect the inhibition of 5-HT-activated current. In addition, 5-HT 3 receptor-mediated currents were inhibited by both 1,9-dideoxy forskolin (30 μM), which does not activate adenylyl cyclase, and wForskolin (30 μM), a charged hydrophilic analogue of forskolin that is membrane impermeable. These results indicate that both forskolin and 17-β-estradiol inhibit the function of the 5-HT 3 receptor in a noncompetitive manner and that this inhibition is independent of cAMP levels.

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria

Prostaglandins (PG) E1, E2and F2 αinduce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1and misoprostol, but blocked the effects of PGF2 αand the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50–500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5–5 μg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.

Adenylate cyclase an potassium channels are involved in forskolin- and 1,9-dideoxyforskolin-induced inhibition of pregnant rat uterus contractility

Objective:We sought to study the contribution of potassium channels in the effect of forskolin and 1,9-dideoxyforskolin on uterine contractility in the pregnant rat. Study Design:Rings taken from the middle portions of uterine horns from rats at 16 days of gestation were positioned in organ chambers containing physiologic salt solution bubbled with 5% carbon dioxide in air (37°C, pH −7.4) for isometric tension recording under 2 g passive tension. The effects of cumulative concentrations of forskolin and 1,9-dideoxyforskolin in the absence or presence of an adenylate cyclase inhibitor (MDL-12,330A, 10−5 mol/L), a nonselective potassium channel blocker (tetrabutylammonium, 10−4 mol/L), or an adenosine triphosphate-dependent potassium channel blocker (glibenclamide 10−5 mol/L) were studied. Results:Both forskolin and, to a lesser extent, 1,9-dideoxyforskolin inhibit uterine contractions. Tetrabutylammonium, glibenclamide, and MDL-12,330A attenuated the effects of forskolin, whereas glibenclamide was less effective against 1,9-dideoxyforskolin. Conclusions:Activation of adenylate cyclases, as well as adenosine triphosphate-dependent potassium channels and, to a greater extent, calcium-dependent potassium channels, is involved in the inhibitory effect of forskolin in uterine rings from rats at 16 days of gestation. Inhibition of uterine contractions by 1,9-dideoxyforskolin is less than that by forskolin and involves activation of adenylate cyclase and calcium-dependent potassium channels. Whether activation of guanylate cyclase is involved in the effect of the agents on calcium-dependent potassium channels needs further investigation. 1,9-Dideoxyforskolin is not an inactive isomer of forskolin in rat uterine rings.

Inhibition of Osteoblast-specific Transcription Factor Cbfa1 by the cAMP Pathway in Osteoblastic Cells: UBIQUITIN/PROTEASOME-DEPENDENT REGULATION

The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function. Parathyroid hormone (through activation of protein kinase A) has also been shown to stimulate ubiquitin/proteasome activity in osteoblasts. Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous treatment with cAMP elevating agents inhibited both osteoblastic differentiation based on alkaline phosphatase assay and DNA binding ability of Cbfa1 based on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibitory effect of forskolin on Cbfa1-regulated genes. Northern and Western blot analyses suggested that the inhibition of Cbfa1 by forskolin was mainly at the protein level. Pretreatment with proteasome inhibitors prior to forskolin treatment reversed the effect of forskolin. Furthermore, addition of proteasome inhibitors to forskolin-pretreated samples resulted in recovery of Cbfa1 protein levels and accumulation of polyubiquitinated forms of Cbfa1, indicating a role for the proteasome pathway in the degradation of Cbfa1. These results suggest that suppression of osteoblastic function by the cAMP pathway is through proteolytic degradation of Cbfa1 involving a ubiquitin/proteasome-dependent mechanism.

Effect of forskolin on endothelin-induced phosphoinositide hydrolysis and calcium mobilization in cultured canine tracheal smooth muscle cells.

1. The effects of increase in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) on endothelin-1 (ET-1)-induced generation of inositol phosphates (IPs) and increase in intracellular Ca2+ ([Ca2+]i) were investigated in canine cultured tracheal smooth muscle cells (TSMCs). 2. Pretreatment of TSMCs with either cholera toxin (CTX; 10 microg ml(-1), 4 h), forskolin (10 microM, 30 min), or dibutyryl cAMP (1 mM, 30 min) inhibited ET-1-stimulated Ca2+ mobilization (by 23 +/- 5%, n = 8) and IPs accumulation (by 32 +/- 6%, n = 4). While after treatment with forskolin for 24 h, the cells retained the ability to respond to ET-1-induced Ca2+ mobilization to the same extent as the control group. 3. Forskolin (1-100 microM) inhibited the ET-1-induced increase in [Ca2+]i, but the lower concentrations had little effect on this response. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration-response curve of ET-1 without changing the -logEC50 values. 4. The water-soluble forskolin analogue L-858051, 7-deacetyl-7beta-(gamma-N-methylpiperazino)-butyryl forskolin, significantly inhibited ET-1-stimulated IPs accumulation. In contrast, the addition of 1,9-dideoxy forskolin, an inactive analogue of forskolin, had little effect on stimulated responses. Moreover, SQ-22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine, an inhibitor of adenylate cyclase, and both H-89, N-(2-aminoethyl)-5-isoquinolinesulfonamide, and HA-1004, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide, inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit ET-1-induced IPs accumulation. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. 5. The locus of this inhibition of forskolin treatment on AlF4(-)-stimulated IPs accumulation was investigated in canine TSMCs. The AlF4(-)-induced IPs accumulation was inhibited by forskolin, supporting that G protein(s) are directly activated by AlF4- and uncoupled to phospholipase C by forskolin treatment. 6. We conclude that cAMP elevating agents inhibit ET-1-stimulated generation of IPs and Ca2+ mobilization in canine cultured TSMCs. Since generation of IPs and increases in [Ca2+]i are very early events in the activation of ET-1 receptors, attenuation of these events by cAMP elevating agents might well contribute to the inhibitory effect of cAMP on tracheal smooth muscle function.

Vascular responsiveness in alloxan-induced diabetes-susceptible (ALS) and resistant (ALR) mice.

Changes in reactivity of vascular smooth muscles of male alloxan-induced diabetes-susceptible (ALS) and resistant (ALR) mice aorta were investigated at 2 weeks, 1, 2 and 4 month(s) after the injection of alloxan (45 mg/kg, i.v.). The glucose levels in blood and urine of all the alloxan-treated ALS mice were markedly elevated while those in alloxan-treated ALR and non-treated ALS and ALR mice were not altered. The magnitude of high K+ (65.4 mM)-induced contractions were not affected by the treatment of alloxan. Norepinephrine-induced contractions in vascular smooth muscles of ALS mice in a diabetic state for 2 or 4 months were significantly potentiated. The contractile sensitivity to prostaglandin F2 alpha (PGF2 alpha) was increased in the 4-month-diabetic state. Responsiveness to 5-HT did not vary in the diabetic mouse. Vasorelaxation induced by nitroprusside was attenuated in 2 weeks, 2 or 4 month-diabetic ALS mice. Similarly the inhibitory effects of levcromakalim were attenuated at 2 and 4 months. The influences of diabetes on the inhibitory effects of forskolin or verapamil were very small or not detected. The effects of the vasomodulators used in this study on the vascular smooth muscles of alloxan-treated ALR mice did not differ from those of untreated ALR mice. The results from using ALS and ALR mice suggest that the vasoreactivities to some vasomodulators are changed especially in the long-term diabetic state and that when diabetes was not induced the dose of alloxan does not have any effect on vascular smooth muscle.

Regulation of PAF-induced platelet responses by cyclic nucleotides

The existence of cross-talk mechanisms between the cyclic nucleotide system and other transduction systems involved in PAF-activated platelets is described in this study. A protein of 125 kDa, identified as pp125FAK, is tyrosine phosphorylated by PAF in a time- and concentration-dependent manner. The presence of a cAMP- or a cGMP-elevating agent, used alone or in combination, together with PAF diminished tyrosine phosphorylation. The sensitivity to cAMP shown by PAF-induced pp125 phosphorylation on tyrosine residues was similar to PAF-induced phosphorylation of a 47-kDa protein (pp47) on serine and threonine. In contrast, the latter was not affected in the presence of a cGMP-elevating agent, although it was able to enhance synergistically the inhibitory effect of forskolin. Data reported herein also show that pp47 phosphorylation and serotonin secretion are not closely correlated. Accordingly, sodium nitroprusside (SNP) did not have any effect on phosphorylation of pp47, but it was able to inhibit serotonin secretion when added alone, and it showed a synergistic inhibitory action with forskolin.

Regulation of PAF-induced platelet responses by cyclic nucleotides

The existence of cross-talk mechanisms between the cyclic nucleotide system and other transduction systems involved in PAF-activated platelets is described in this study. A protein of 125 kDa, identified as pp 125FAK, is tyrosine phosphorylated by PAF in a time- and concentration-dependent manner. The presence of a cAMP- or a cGMP-elevating agent, used alone or in combination, together with PAF diminished tyrosine phosphorylation. The sensitivity to cAMP shown by PAF-induced ppl25 phosphorylation on tyrosine residues was similar to PAF-induced phosphorylation of a 47-kDa protein (pp47) on serine and threonine. In contrast, the latter was not affected in the presence of a cGMP-elevating agent, although it was able to enhance synergistically the inhibitory effect of forskolin. Data reported herein also show that pp47 phosphorylation and serotonin secretion are not closely correlated. Accordingly, sodium nitroprusside (SNP) did not have any effect on phosphorylation of pp47, but it was able to inhibit serotonin secretion when added alone, and it showed a synergistic inhibitory action with forskolin.

Mutations to forskolin resistance result in loss of adrenocorticotropin receptors and consequent reductions in levels of G protein alpha-subunits.

A family of mutants isolated from the Y1 mouse adrenal cell line on the basis of their resistance to the growth inhibitory effects of forskolin have an underlying mutation that affects the activity of adenylyl cyclase. As part of the mutant phenotype, adenylyl cyclase is partially resistant to activation by forskolin, completely insensitive to ACTH, and fully responsive to NaF; the levels of Gs alpha and G1 alpha in plasma membrane fractions are decreased; and the activity of G beta/gamma is impaired. In the present study, we examine the basis for the complex phenotype associated with forskolin resistance to better understand the factors that contribute to the regulation of adenylyl cyclase activity. We demonstrate that the resistance of these mutants to ACTH results from the failure to express ACTH receptor transcripts. Transfection of these mutants with a gene encoding the mouse beta 2-adrenergic receptor led to the recovery of transformants with normal receptor-G protein coupling and with increased levels of Gs alpha and G1 alpha that approached those in parental Y1 cells. These beta 2-adrenergic receptor transformants, nonetheless, remained resistant to forskolin and ACTH. Two spontaneous Y1 mutants, Y6 and OS3, previously characterized as ACTH-resistant clones that failed to accumulate ACTH receptor transcripts, were shown to be forskolin resistant and to contain less Gs alpha in membrane fractions, indicating that forskolin resistance, failure to express the ACTH receptor, and the consequent reduction in Gs alpha are closely linked. Expression of the human ACTH receptor in Y6 and OS3 cells restored ACTH-responsive adenylyl cyclase activity and increased the level of Gs alpha, but did not otherwise reverse the forskolin-resistant phenotype. Together, these results demonstrate that mutations to forskolin resistance have downstream consequences that result in the loss of ACTH receptor expression and the consequent reduction in levels of membrane-associated G alpha subunits. The results further suggest that G protein-coupled receptors may have a stabilizing influence on G alpha subunits associated with the cell membrane. According to current models, forskolin activates adenylyl cyclase by forming a ternary complex with adenylyl cyclase and Gs alpha. Our results suggest that this model may be incomplete and that an additional component, acting directly or indirectly, is required for optimal activation of adenylyl cyclase by forskolin.