Forskolin-induced Decrease Research Articles

Different rate-limiting activities of intracellular pH regulators for HCO3- secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts.

Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3- release from HCO3--secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na+-H+ exchanger (NHE) to pHi regulation for HCO3- secretion by cAMP and Ca2+ signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca2+-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3- secretion.

Regulation of anion transport across the uterine epithelium ofGallus domesticus

For egg shell production, cations, primarily Ca(2+), and anions, primarily HCO(3)(-), must be secreted across the uterine epithelium. Because alterations of HCO(3)(-) transport influence Ca(2+) secretion, the present study was performed to gain insight into the regulation of anion transport (i.e., chloride and HCO(3)(-) transport) across the egg shell gland of the domestic chicken. To this purpose, unstripped uterus epithelia were mounted in modified Ussing chambers and electrogenic [i.e., short circuit current (I(sc))] and electroneutral anion transport were measured. Stimulating adenylate cyclases by forskolin, thereby enhancing the intracellular cyclic adenosine monophosphate( )(cAMP) concentration, evoked 2 patterns of I(sc) responses. Under HCO(3)(-)-buffered conditions, some of the tissues (3/8) showed an increase of I(sc), whereas in others (5/8) a decrease of I(sc) was observed in the presence of the drug. The I(sc) increase existed in HCO(3)(-) secretion because under HCO(3)(-)-free conditions forskolin stimulated only an I(sc) decrease. In addition, methazolamide, a blocker of carboanhydrases, significantly reduced baseline I(sc). The forskolin-induced decrease of I(sc) presented a Cl(-) absorption. In the absence of HCO(3)(-), forskolin yielded only an I(sc) decrease and the transepithelial flux of (36)Cl(-) was reduced. In the presence of mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid, a nonselective blocker of chloride channels, forskolin-induced I(sc) decrease was inhibited. In addition to cAMP, the regulatory influence of neurons or prostaglandins on baseline I(sc) and anion transport was investigated. Neither tetrodotoxin, a blocker of neuronal Na(+) conductances, electrical field stimulation, nor indomethacin, a blocker of cyclooxygenases, influenced the baseline I(sc) or anion transport. In summary, these data show effects of forskolin (i.e., cAMP) on anion transport across the egg shell gland of the domestic chicken: HCO(3)(-) secretion (supporting Ca(2+) mineralizing of the egg shell) and Cl(-) absorption. Neurons or prostaglandins did not influence the anion transport.

Activation of cAMP-dependent Signaling Induces Oxidative Modification of the Cardiac Na+-K+ Pump and Inhibits Its Activity

Cellular signaling can inhibit the membrane Na(+)-K(+) pump via protein kinase C (PKC)-dependent activation of NADPH oxidase and a downstream oxidative modification, glutathionylation, of the beta(1) subunit of the pump alpha/beta heterodimer. It is firmly established that cAMP-dependent signaling also regulates the pump, and we have now examined the hypothesis that such regulation can be mediated by glutathionylation. Exposure of rabbit cardiac myocytes to the adenylyl cyclase activator forskolin increased the co-immunoprecipitation of NADPH oxidase subunits p47(phox) and p22(phox), required for its activation, and increased superoxide-sensitive fluorescence. Forskolin also increased glutathionylation of the Na(+)-K(+) pump beta(1) subunit and decreased its co-immunoprecipitation with the alpha(1) subunit, findings similar to those already established for PKC-dependent signaling. The decrease in co-immunoprecipitation indicates a decrease in the alpha(1)/beta(1) subunit interaction known to be critical for pump function. In agreement with this, forskolin decreased ouabain-sensitive electrogenic Na(+)-K(+) pump current (arising from the 3:2 Na(+):K(+) exchange ratio) of voltage-clamped, internally perfused myocytes. The decrease was abolished by the inclusion of superoxide dismutase, the inhibitory peptide for the epsilon-isoform of PKC or inhibitory peptide for NADPH oxidase in patch pipette solutions that perfuse the intracellular compartment. Pump inhibition was also abolished by inhibitors of protein kinase A and phospholipase C. We conclude that cAMP- and PKC-dependent inhibition of the cardiac Na(+)-K(+) pump occurs via a shared downstream oxidative signaling pathway involving NADPH oxidase activation and glutathionylation of the pump beta(1) subunit.

Contribution of CFTR to apical-basolateral fluid transport in cultured human alveolar epithelial type II cells

Previous studies in intact lung suggest that CFTR may play a role in cAMP-regulated fluid transport from the distal air spaces of the lung. However, the potential contribution of different epithelial cells (alveolar epithelial type I, type II, or bronchial epithelial cells) to CFTR-regulated fluid transport is unknown. In this study we determined whether the CFTR gene is expressed in human lung alveolar epithelial type II (AT II) cells and whether the CFTR chloride channel contributes to cAMP-regulated fluid transport in cultured human AT II cells. Human AT II cells were isolated and cultured on collagen I-coated Transwell membranes for 120-144 h with an air-liquid interface. The cultured cells retained typical AT II-like features based on morphologic studies. Net basal fluid transport was 0.9+/-0.1 mul.cm-2.h-1 and increased to 1.35+/-0.11 mul.cm-2.h-1 (mean+/-SE, n=18, P<0.05) by stimulation with cAMP agonists. The CFTR inhibitor, CFTRinh-172, inhibited cAMP stimulated but not basal fluid transport. In short-circuit current (Isc) studies with an apical-to-basolateral transepithelial Cl- gradient, apical application of CFTRinh-172 reversed the forskolin-induced decrease in Isc. Real time RT-PCR demonstrated CFTR transcript expression in human AT II cells at a level similar to that in airway epithelial cells. We conclude that CFTR is expressed in cultured human AT II cells and may contribute to cAMP-regulated apical-basolateral fluid transport.

Forskolin-induced clearance of the fluorescent dye sulforhodamine from rat parotid intralobular duct lumen: visualization of the secretory function under a confocal laser scanning microscope.

Cyclic AMP evokes fluid secretion with bicarbonate in exocrine ducts. Clearance of fluorescent dyes from rat parotid intralobular ducts by forskolin was visualized as a fluorescence change in the duct luminal space by optical sectioning under a confocal laser scanning microscope to clarify the secretory function in the ducts. When the isolated rat parotid intralobular duct segments were superfused with membrane-impermeable fluorescent dyes during the experimental period, fluorescent dyes were passively moved into the duct space. Forskolin and isobutylmethylxanthine decreased the fluorescence of anionic dye, sulforhodamine B, and neutral dye, dextran tetramethyl-rhodamine, in the duct space, suggesting that the forskolin-induced clearance of fluorescent dyes might be the result of fluid secretion in the ducts. Methazolamide inhibited a forskolin-induced sustained decrease in duct fluorescence and intracellular acidification. Low concentrations of external Cl?, DIDS, bumetanide and amiloride did not markedly inhibit a forskolin-induced decrease in duct fluorescence. These findings suggest that a major portion of the steady decrease in duct fluorescence by forskolin was related to intracellular HCO3? production, not the uptake mechanism of external Cl?. Glibenclamide, NPPB, DPC and DMA inhibited the forskolin-induced decrease. Forskolin evokes the clearance of fluorescent dyes from duct space possibly due to fluid secretion in rat parotid ducts, associated with secretion through CFTR and DPC-sensitive anion channels of carbonic anhydrase-dependent bicarbonate linked with the Na+/H+ exchange mechanism.

Regulation of RGS mRNAs by cAMP in PC12 Cells

The RGS (regulators of G protein signaling) proteins represent a novel family of proteins which attenuate G protein mediated signaling. Using antisense riboprobes selective for rat RGS4, RGS7, and RGS2, we examined the regulation of these RGS mRNAs in PC12 cells in response to agents which elevate intracellular cAMP. Treatment of the PC12 cells with forskolin, dibutryl cAMP, or 8-CPT-cAMP for three hours decreased RGS4 message by nearly 50%. Actinomycin D, a potent inhibitor of transcription, did not affect the forskolin-induced decrease in RGS4 message, suggesting that forskolin does not alter RGS4 message half-life. RGS7 message is also present in these cells, but was not affected by forskolin. In contrast, RGS2 message is not evident in unstimulated cells but is strongly induced by one hour of treatment with forskolin. Taken together, these data suggest that mRNA levels of different RGS family members respond in an idiosynchratic fashion to cAMP challenge.

Cyclic AMP-dependent pathway that mediates suppressive regulation of glycosaminoglycan production in cultured vascular endothelial cells

We investigated the effect of agents which increase the intracellular level of cyclic AMP (cAMP) on the production of glycosaminoglycans (GAGs) by cultured bovine aortic endothelial cells. It was found that the incorporation of [ 3H]glucosamine into GAGs was significantly decreased by forskolin, 8-bromo cAMP, 3-isobutyl-1-methylxanthine and prostaglandin E 1. The leakage of lactate dehydrogenase into the medium, a marker of nonspecific cell damage, were not changed by forskolin. Forskolin-induced decrease in the [ 3H]glucosamine incorporation occurred in both heparan sulfate and the other GAGs. The [ 3H]glucosamine incorporation into GAGs was suppressed by forskolin also in human aortic and human umbilical vein endothelial cells, bovine aortic smooth muscle cells, porcine kidney epithelial LLC-PK 1 cells and human fetal lung fibroblastic IMR-90 cells. In conclusion, it was suggested that intracellular cAMP mediates the suppressive regulation of GAG production in vascular endothelial cells; as a result, the cell surface heparan sulfate was reduced. The cAMP-dependent pathway appears to be important as an intracellular mechanism by which endothelial production of anticoagulant heparan sulfate is regulated.

Transcriptional regulation of CREB (cyclic AMP response element-binding protein) expression in CATH.a cells.

We have recently demonstrated that mRNA expression of cyclic AMP (cAMP) response element-binding protein (CREB) is down-regulated in CATH.a cells (a neural-derived cell line) by activation of the cAMP pathway. We now demonstrate that this down-regulation can be accounted for by a decrease in the rate of CREB gene transcription. It was found that cycloheximide, a protein synthesis inhibitor, prevented the forskolin-induced decrease in CREB mRNA levels in CATH.a cells. Nuclear run-on assays demonstrated that forskolin decreased the rate of CREB transcription by close to 50%. Moreover, forskolin decreased chloramphenicol acetyltransferase (CAT) activity in CATH.a cells transiently transfected with a construct containing 1,240 bp of CREB promoter fused to a CAT reporter plasmid. Possible mechanisms by which activation of the cAMP pathway leads to a decrease in CREB gene transcription are discussed.

Effects of adenylyl cyclase-linked neuropeptides on the expression of ciliary neurotrophic factor-mRNA in cultured astrocytes

Ciliary neurotrophic factor (CNTF) is a molecule which has profound effects on various neural cell types. In the central nervous system, expression of CNTF-mRNA is highly concentrated in olfactory bulb. In the present study, we examined the regulatory mechanism of CNTF-mRNA expression in cultured astrocytes from newborn rat brain. Cultured astrocytes from new born rat brain expressed CNTF-mRNA at levels comparable to the level in olfactory bulb in vivo. Treatment of the astrocytes with forskolin, an activator of adenylyl cyclase, led to a decrease of CNTF-mRNA level. The effect of forskolin was mimicked by cAMP-linked agonists, such as VIP, PACAP, isoproterenol and dopamine. Cycloheximide, an inhibitor of protein synthesis, did not abolish the forskolin-induced decrease of CNTF-mRNA. Measurement of the half-life of CNTF-mRNA in the presence of actinomycin D, an inhibitor of transcription, indicated that the degradation of CNTF-mRNA is not destabilized by the forskolin-treatment. These data taken together suggest that the cAMP-induced suppression of CNTF-mRNA is mainly caused by the inhibition of CNTF gene transcription.

Forskolin inhibits platelet-activating factor binding to platelet receptors independently of adenylyl cyclase activation

The effect of forskolin on platelet-activating factor (PAF) receptor was investigated. Rabbit platelets treated with forskolin showed approximately a 9-fold increase in cAMP levels over the control. After treatment of platelets with forskolin prior to PAF binding, a 30–40% (P < 0.005) decrease in PAF binding was observed. The decrease in PAF binding caused by forskolin was concomitant with a decrease in the physiological responses of platelets induced by PAF. However, this forskolin-induced decrease in PAF binding was not a consequence of cAMP formation as the addition of a cAMP analog could not mimic the action of forskolin. Additionally, the inactive analog of forskolin, dideoxyforskolin, which does not activate adenylyl cyclase, also reduced PAF binding to its receptor. Reduction of PAF binding by forskolin and dideoxyforskolin was also observed with isolated platelet membranes. To understand the mechanism of forskolin induced changes in PAF binding, the involvement of a G-protein in this process was investigated. Cells treated with GTPγS showed approximately a 25% reduction in PAF binding. Addition of forskolin to the GTPγS treated cells resulted in a further reduction in PAF binding, suggesting the action of forskolin was independent of G-protein activation. The data suggests that the action of forskolin was independent of adenylyl cyclase or G-protein involvement. It is speculated that the action of forskolin on PAF binding was due to a direct effect of this molecule and its analog on the PAF receptor itself or to components of the post-receptor signalling for PAF.

Effects of Adenylate Cyclase Activation on Electrical Resistance of Scala Media

Electrical resistance of scala media at the second turn of the guinea pig cochlea was measured while cochlear adenylate cyclase was activated by perilymphatic perfusion with 2 x 10(-4) M forskolin. The electrical resistance decreased from the pre-forskolin value (6.36 +/- 0.27 k omega to 5.22 +/- 0.33 k omega, n = 5), while the endocochlear potential (EP) increased from the pre-forskolin value (70.2 +/- 2.1 mV to 81.2 +/- 2.8 mV, n = 5). The time course of the electrical resistance change was more rapid than that of the EP change but almost identical to the time course of the change in the Cl- activity of the endolymph. The results suggest that the forskolin-induced decrease in the electrical resistance of the perilymph-endolymph barrier (presumably Reissner's membrane), and the consequent increase in Cl- flux into the endolymph may trigger EP elevation.

Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells. Evidence for increased degradation of catalytic subunit in the presence of cAMP.

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.

Phorbol ester prevents the thyroid‐stimulating‐hormone‐induced but not the forskolin‐induced decrease of cAMP‐dependent protein kinase activity in thyroid cell cultures

The potent tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate (TPA) affects several thyroid cell functions and interacts with thyroid-stimulating hormone (TSH) either by inhibiting or potentiating its action on different cellular parameters. Since phorbol ester acts mainly through the activation of protein kinase C, which is its receptor, we studied this activation and its interaction with TSH and forskolin in suspension cultures of porcine thyroid cells. In thyroid cell cultures, TPA has a dual effect on protein kinase C activity: immediately (2-5 min) after exposure of cells to TPA, it began to be translocated from the cytosol to the particulate fraction. The transfer of the cytosolic enzyme was total and could occur with or without a loss of activity. The translocated enzyme still needed Ca2+ and phospholipids for its activation. The basal activity increased transiently (2-4 h) in both the cytosol and particulate fractions during translocation. The peak activity in the particulate fraction was reached 10-30 min after exposure of cells to TPA, and was followed by down-regulation of protein kinase C and almost complete disappearance of its activity. The residual activity was about 13% of control after a 2-day exposure to TPA. It was unequally distributed between cytosol (4%) and particulate fraction (9%). Prolonged exposure of cells to TPA did not affect either the activity or the subcellular distribution of the cAMP-dependent protein kinase activity. TPA interacted with TSH and prevented the decrease of this activity induced by prolonged exposure of cells to the hormone not only when it was introduced simultaneously with TSH, but also when it was added 24 h after TSH. However, the forskolin-induced decrease in cAMP-dependent protein kinase activity was not prevented by the presence of TPA. TPA also affected the increases in cAMP accumulation mediated by TSH and forskolin. The TSH-induced increase was significantly stimulated by TPA after short contacts (5-15 min), while longer preincubations of cells with TPA provoked a very strong inhibition of the TSH action. However, the forskolin-induced stimulation of the cAMP accumulation was maintained and even further increased in the presence of TPA. Consequently, the actions of TSH and TPA are apparently interdependent, while those of forskolin and TPA seem to be parallel and independent. Neither TSH nor forskolin prevented the TPA-induced down regulation of protein kinase C. The biologically inactive phorbol ester analogue 4 alpha-phorbol 12,13-didecanoate had no effect on protein kinase C activity, and did not interact with either TSH or forskolin.(ABSTRACT TRUNCATED AT 400 WORDS)