microM Forskolin Research Articles

Basolateral anion transport mechanisms underlying fluid secretion by mouse, rat and guinea-pig pancreatic ducts.

Fluid secretion by interlobular pancreatic ducts was determined by using video microscopy to measure the rate of swelling of isolated duct segments that had sealed following overnight culture. The aim was to compare the HCO(3)(-) requirement for secretin-evoked secretion in mouse, rat and guinea-pig pancreas. In mouse and rat ducts, fluid secretion could be evoked by 10 nm secretin and 5 microm forskolin in the absence of extracellular HCO(3)(-). In guinea-pig ducts, however, fluid secretion was totally dependent on HCO(3)(-). Forskolin-stimulated fluid secretion by mouse and rat ducts in the absence of HCO(3)(-) was dependent on extracellular Cl(-) and was completely inhibited by bumetanide (30 microm). It was therefore probably mediated by a basolateral Na(+)-K(+)-2Cl(-) cotransporter. In the presence of HCO(3)(-), forskolin-stimulated fluid secretion was reduced approximately 40% by bumetanide, approximately 50% by inhibitors of basolateral HCO(3)(-) uptake (3 microm EIPA and 500 microm H(2)DIDS), and was totally abolished by simultaneous application of all three inhibitors. We conclude that the driving force for secretin-evoked fluid secretion by mouse and rat ducts is provided by parallel basolateral mechanisms: Na(+)-H(+) exchange and Na(+)-HCO(3)(-) cotransport mediating HCO(3)(-) uptake, and Na(+)-K(+)-2Cl(-) cotransport mediating Cl(-) uptake. The absence or inactivity of the Cl(-) uptake pathway in the guinea-pig pancreatic ducts may help to account for the much higher concentrations of HCO(3)(-) secreted in this species.

Regulation of L-type calcium current by intracellular magnesium in rat cardiac myocytes.

The effects of changing cytosolic [Mg(2+)] ([Mg(2+)](i)) on L-type Ca(2+) currents were investigated in rat cardiac ventricular myocytes voltage-clamped with patch pipettes containing salt solutions with defined [Mg(2+)] and [Ca(2+)]. To control [Mg(2+)](i) and cytosolic [Ca(2+)] ([Ca(2+)](i)), the pipette solution included 30 mM citrate and 10 mM ATP along with 5 mM EGTA (slow Ca(2+) buffer) or 15 mM EGTA plus 5 mM BAPTA (fast Ca(2+) buffer). With pipette [Ca(2+)] ([Ca(2+)](p)) set at 100 nM using a slow Ca(2+) buffer and pipette [Mg(2+)] ([Mg(2+)](p)) set at 0.2 mM, peak l-type Ca(2+) current density (I(Ca)) was 17.0 +/- 2.2 pA pF(-1). Under the same conditions, but with [Mg(2+)](p) set to 1.8 mM, I(Ca) was 5.6 +/- 1.0 pA pF(-1), a 64 +/- 2.8% decrease in amplitude. This decrease in I(Ca) was accompanied by an acceleration and a -8 mV shift in the voltage dependence of current inactivation. The [Mg(2+)](p)-dependent decrease in I(Ca) was not significantly different when myocytes were preincubated with 10 microM forskolin and 300 microM 3-isobutyl-L-methylxanthine and voltage-clamped with pipettes containing 50 microM okadaic acid, to maximize Ca(2+) channel phosphorylation. However, when myocytes were voltage-clamped with pipettes containing protein phosphatase 2A, to promote channel dephosphorylation, I(Ca) decreased only 25 +/- 3.4% on changing [Mg(2+)](p) from 0.2 to 1.8 mM. In the presence of 0.2 mM[Mg(2+)](p), changing channel phosphorylation conditions altered I(Ca) over a 4-fold range; however, with 1.8 mM[Mg(2+)](p), these same manoeuvres had a much smaller effect on I(Ca). These data suggest that [Mg(2+)](i) can antagonize the effects of phosphorylation on channel gating kinetics. Setting [Ca(2+)](p) to 1, 100 or 300 nM also showed that the [Mg(2+)](p)-induced reduction of I(Ca) was smaller at the lowest [Ca(2+)](p), irrespective of channel phosphorylation conditions. This interaction between [Ca(2+)](i) and [Mg(2+)](i) to modulate I(Ca) was not significantly affected by ryanodine, fast Ca(2+) buffers or inhibitors of calmodulin, calmodulin-dependent kinase and calcineurin. Thus, physiologically relevant [Mg(2+)](i) modulates I(Ca) by counteracting the effects of Ca(2+) channel phosphorylation and by an unknown [Ca(2+)](i)-dependent mechanism. The magnitude of these effects suggests that changes in [Mg(2+)](i) could be critical in regulating L-type channel gating.

HCO3-]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

BackgroundBicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO3- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl- permeability. Here, we examined whether HCO3- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO3- and the stimulation of CFTR in BCECs.ResultsRT-competitive PCR indicated that sAC mRNA expression in BCECs is dependent on [HCO3-] and incubation time in HCO3-. Immunoblots showed that 10 and 40 mM HCO3- increased sAC protein expression by 45% and 87%, respectively, relative to cells cultured in the absence of HCO3-. Furthermore, 40 mM HCO3- up-regulated sAC protein expression in Calu-3 cells by 93%. On the other hand, sAC expression in BCECs and Calu-3 cells was unaffected by changes in bath pH or osmolarity. Interestingly, BCECs pre-treated with10 μM adenosine or 10 μM forskolin, which increase cAMP levels, showed decreased sAC mRNA expression by 20% and 30%, respectively. Intracellular cAMP production by sAC paralleled the time and [HCO3-]-dependent expression of sAC. Bicarbonate-induced apical Cl- permeability increased by 78% (P < 0.01) in BCECs cultured in HCO3-. However for cells cultured in the absence of HCO3-, apical Cl- permeability increased by only 10.3% (P > 0.05).ConclusionHCO3- not only directly activates sAC, but also up-regulates the expression of sAC. These results suggest that active cellular uptake of HCO3- can contribute to the basal level of cellular cAMP in tissues that express sAC.

Direct, rapid effects of 25-hydroxyvitamin D3 on isolated intestinal cells.

Scattered reports in the literature have suggested that the metabolite 25-hydroxyvitamin D(3) [25(OH)D(3)] has biological activity. In the present work, perfusion of isolated duodenal loops of normal chickens with 100 nM 25(OH)D(3) resulted in enhanced transport of (45)Ca within 2 min relative to the vehicle controls. We then tested the effect of a range of 25(OH)D(3) concentrations on (45)Ca handling by isolated intestinal cells in time course studies. Following a basal uptake period, cell suspensions from 7-week old chicks were treated either with 25, 100, or 300 nM 25(OH)D(3), or the vehicle ethanol (0.01%, final concentration). Both 25 and 100 nM 25(OH)D(3) resulted in a significant (P < 0.05) reduction in (45)Ca levels, relative to controls, between 1-10 min after treatment, while 300 nM 25(OH)D(3) resulted in a significant increase in (45)Ca levels, relative to controls, after 10 min of incubation. The effect of 100 nM 25(OH)D(3) (a physiological level) on cell calcium was abolished by the presence of 6.5 nM 24,25-dihydroxyvitamin D(3). In cell preparations from 14- or 28-week old birds 100nM 25(OH)D(3) had no effect, relative to vehicle controls. Incubation of cells with 2 microM BAY K8644, a calcium channel activator, stimulated (45)Ca uptake within 3 min relative to vehicle controls (P < 0.05), while addition of either 20 microM forskolin or 100 nM phorbol ester (stimulators of the PKA and PKC pathways, respectively) resulted in enhanced radionuclide levels after 10 min of incubation (P < 0.05, relative to corresponding controls). Finally, cells were treated with 100 nM 25(OH)D(3) or vehicle and samples taken at various times for analyses of protein kinase C and A activities. No effect of 25(OH)D(3) on protein kinase C activity was observed, while protein kinase A activity was stimulated to nearly 200% of controls at 1 min after 25(OH)D(3) addition (P < 0.05, relative to corresponding controls) and began declining at 3 min, returning to control levels 5 min after additions. We conclude that 25(OH)D(3) has a direct effect on calcium handling in enterocytes of young animals that may in part be mediated by the protein kinase A signal transduction pathway.

Intracellular Ca(2+) regulates responsiveness of cardiac L-type Ca(2+) current to protein kinase A: role of calmodulin.

The goal of this study was to determine whether the protein kinase A (PKA) responsiveness of the cardiac L-type Ca(2+) current (ICa) is affected during transient increases in intracellular Ca(2+) concentration. Ventricular myocytes were isolated from 3- to 4-day-old neonatal rats and cultured on aligned collagen thin gels. When measured in 1 or 2 mM Ca(2+) external solution, the aligned myocytes displayed a large ICa that was weakly regulated (20% increase) during stimulation of PKA by 2 microM forskolin. In contrast, application of forskolin caused a 100% increase in ICa when the external Ca(2+) concentration was reduced to 0.5 mM or replaced with Ba(2+). This Ca(2+)-dependent inhibition was also observed when the cells were treated with 1 microM isoproterenol, 100 microM 3-isobutyl-1-methylxanthine, or 500 microM 8-bromo-cAMP. The responsiveness of ICa to PKA was restored during intracellular dialysis with a calmodulin (CaM) inhibitory peptide but not during treatment with inhibitors of protein kinase C, Ca(2+)/CaM-dependent protein kinase, or calcineurin. Adenoviral-mediated expression of a CaM molecule with mutations in all four Ca(2+)-binding sites also increased the PKA sensitivity of ICa. Finally, adult mouse ventricular myocytes displayed a greater response to forskolin and cAMP in external Ba(2+). Thus Ca(2+) entering the myocyte through the voltage-gated Ca(2+) channel regulates the PKA responsiveness of ICa.

Angiotensin II activates cholesterol ester hydrolase in bovine adrenal glomerulosa cells through phosphorylation mediated by p42/p44 mitogen-activated protein kinase.

In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) occurs via activation of the Ca2+ messenger system, increased expression of the steroidogenic acute regulatory protein, and enhanced transfer of cholesterol to the inner mitochondrial membrane. We examined here whether Ang II affects the activity of cholesterol ester hydrolase (CEH), also named hormone-sensitive lipase, the enzyme recruiting cholesterol from intracellular pools, in bovine adrenal glomerulosa cells. In bovine adrenal tissue, CEH activity was detected with characteristics similar to those reported in other tissues (Michaelis constant = 46.3 +/- 6.7 microM, n = 3; maximal velocity = 1 nmol/mg.min). This activity was significantly enhanced in isolated bovine glomerulosa cells challenged for 2 h with 10 nM Ang II (to 149 +/- 11% of controls, n = 3). Similarly, 25 microM forskolin raised CEH activity to 151 +/- 5% of controls (n = 3). This increase in activity of CEH was not due to an increase in the amount of enzyme protein but was associated with an increased phosphorylation of the enzyme to 337 +/- 33% of controls (n = 9, P < 0.0001). Potassium ion (K+) and forskolin also stimulated [32P]orthophosphate incorporation, although to a lesser extent (to 157 +/- 18% and 186 +/- 25% of controls, respectively). On SDS-PAGE, the majority of this radioactivity was associated with a species of 172 kDa, corresponding to a CEH dimer. Both Ang II-induced CEH phosphorylation and pregnenolone production were significantly reduced (to 47 +/- 6% and 50 +/- 8% of controls with Ang II alone, respectively) in the presence of PD098059, an inhibitor of p42/p44 MAPK. Indeed, Ang II challenge led to a rapid 32P incorporation into p42/p44 MAPK. These results demonstrate that, in addition to its known effects on intramitochondrial cholesterol transfer, Ang II also promotes aldosterone biosynthesis by rapidly increasing cholesterol supply to the outer mitochondrial membrane.

Targeting of Protein Kinase A by Muscle A Kinase-anchoring Protein (mAKAP) Regulates Phosphorylation and Function of the Skeletal Muscle Ryanodine Receptor

Protein kinase A anchoring proteins (AKAPs) tether cAMP-dependent protein kinase (PKA) to specific subcellular locations. The muscle AKAP, mAKAP, co-localizes with the sarcoplasmic reticulum Ca2+ release channel or ryanodine receptor (RyR). The purpose of this study was to determine whether anchoring of PKA by mAKAP regulates RyR function. Either mAKAP or mAKAP-P, which is unable to anchor PKA, was expressed in CHO cells stably expressing the skeletal muscle isoform of RyR (CHO-RyR1). Immunoelectron microscopy showed that mAKAP co-localized with RyR1 in disrupted skeletal muscle. Following the addition of 10 microm forskolin to activate adenylyl cyclase, RyR1 phosphorylation in CHO-RyR1 cells expressing mAKAP increased by 42.4 +/- 6.6% (n = 4) compared with cells expressing mAKAP-P. Forskolin treatment alone did not increase the amplitude of the cytosolic Ca2+ transient in CHO-RyR1 cells expressing mAKAP or mAKAP-P; however, forskolin plus 10 mm caffeine elicited a cytosolic Ca2+ transient, the amplitude of which increased by 22% (p < 0.05) in RyR1/mAKAP-expressing cells compared with RyR1/mAKAP-P-expressing cells. Therefore, localization of PKA by mAKAP at RyR1 increases both PKA-dependent RyR phosphorylation as well as efflux of Ca2+ through the RyR. Therefore, RyR1 function is regulated by mAKAP targeting of PKA, implying an important functional role for PKA phosphorylation of RyR in skeletal muscle.

Inhibitory effects of interferon-gamma on the heterologous desensitization of beta-adrenoceptors by transforming growth factor-beta 1 in tracheal smooth muscle.

Transforming growth factor-beta1 (TGF-beta 1) is generally considered to play an important role in the pathogenesis of chronic inflammation and fibrosis. This study was designed to determine mechanisms of reduced responsiveness of guinea-pig tracheal smooth muscle to beta-adrenoceptor agonists by TGF-beta 1, using isometric tension records and tissue cAMP measurement. Moreover, we examined the involvement of the signal transduction processes of TGF-beta superfamily in the desensitization of beta-adrenoceptors. After exposure to 0.2-2000 pm TGF-beta 1 for 4-8 h, the inhibitory effects of 1 microm isoprenaline (ISO) and 10 microm forskolin on 1 microm MCh-induced contraction were markedly reduced in a concentration-dependent fashion. The desensitization by TGF-beta 1 was greater against ISO than for forskolin. The values of EC75 for the curves for ISO after exposure to the normal bathing solution and TGF-beta 1 were 0.039 +/- 0.02 and 0.38 +/- 0.28 microm, respectively. The values of EC50 for the curves for forskolin under these conditions were 0.50 +/- 0.12 and 0.89 +/- 0.21 microm, respectively. On the other hand, the inhibitory effects of phosphodiesterase inhibitors such as theophylline and rolipram were not attenuated after exposure to TGF-beta 1. Concentration-inhibition curve for ISO was shifted to the right after exposure to 2000 pm TGF-beta 1 for 8 h more than that curve for forskolin. In contrast, the curve for theophylline was not shifted to the right by TGF-beta 1. When the tissues were incubated with TGF-beta 1 in the presence of IFN-gamma, an intracellular antagonist of TGF-beta signalling, IFN-gamma inhibited the reduced response to ISO and forskolin after exposure to TGF-beta 1 in a concentration-dependent fashion. After exposure to TGF-beta 1, the effects of cAMP accumulation of ISO was significantly reduced, however, neither forskolin-nor theophylline-induced cAMP accumulation was affected. IFN-gamma had no significant effect on cAMP accumulation either to ISO or forskolin. Impairment of the beta-adrenoceptors/adenylyl cyclase pathway are involved in heterologous desensitization of beta-adrenoceptors induced by TGF-beta 1 in airway smooth muscle. IFN-gamma functionally suppresses this phenomenon via cAMP-independent processes. Phosphodiesterase is still intact under this condition.

EGF stimulates growth by enhancing capacitative calcium entry in corneal epithelial cells.

In rabbit corneal epithelial cells (RCEC), we determined whether capacitative calcium entry (CCE) mediates the mitogenic response to epidermal growth factor, EGF. [Ca2+]i was measured with single-cell fluorescence imaging of fura2-loaded RCEC. EGF (5 ng/ml) maximally increased [Ca2+]i 4.4-fold. Following intracellular store (ICS) calcium depletion in calcium-free medium with 10 microM cyclopiazonic acid (CPA) (endoplasmic reticulum calcium ATPase inhibitor), calcium addback elicited plasma membrane Ca2+ influx as a result of activation of plasma membrane store operated channel (SOC) activity. Based on Mn2+ quench measurements of fura2 fluorescence, 5 ng/ml EGF enhanced such influx 2.3-fold, whereas with Rp-cAMPS (protein kinase A inhibitor) plus EGF it increased by 5.3-fold. In contrast, SOC activation was blocked with 100 microM 2-aminoethyldiphenylborate (2-APB, store-operated channel inhibitor). During exposure to either 50 microM UO126 (MEK-1/2 inhibitor) or 10 microM forskolin (adenylate cyclase activator), 5 ng/ml EGF failed to affect [Ca2+]i. RT-PCR detected gene expression of: 1) transient receptor potential (TRP) protein isoforms 1, 3, 4, 6 and 7; 2) IP3R isoforms 1-3. Immunocytochemistry, in conjunction with confocal and immunogold electron microscopy, detected plasma membrane localization of TRP4 expression. Inhibition of CCE with 2-APB and/or CPA, eliminated the 2.5-fold increase in intracellular [3H]-thymidine incorporation induced by EGF. Taken together, CCE in RCEC mediates the mitogenic response to EGF. EGF induces CCE through its stimulation of Erkl/2 activity, whereas PKA stimulation suppresses these effects of EGF. TRP4 may be a component of plasma membrane SOC activity, which is stimulated by ICS calcium depletion.

Involvement of Ca2+ mobilization in tachyphylaxis to beta-adrenergic receptors in trachealis.

We examined the mechanisms underlying tachyphylaxis to beta-adrenergic receptor agonists (beta-agonists) in tracheal smooth muscle. Simultaneous measurements of isometric tension and intracellular Ca2+ concentration ([Ca2+]i) using fura-2-loaded guinea pig tracheas showed that the inhibitory effects of isoproterenol (ISO) on tension and increases in [Ca2+]i induced by methacholine exhibited marked tachyphylaxis with repeated exposure to ISO at intervals of 15 min. Similarly, the activation of single Ca2+-activated K+ (KCa) channels in on-cell patches by 1 microM ISO was gradually attenuated after repeated extracellular application of ISO to single smooth cells of porcine tracheas. Desensitization of beta-adrenergic receptor/KCa channel stimulatory coupling and relaxation responses was prevented by separately antagonizing the voltage-dependent Ca2+ channel (VDCC) with verapamil, suggesting a surprising relationship between Ca2+ influx through VDCC and beta-adrenergic desensitization. Conversely, repeated exposure of 10 U/ml protein kinase A to inside-out patches did not result in desensitization of channel activation, and repeated exposure to 10 microM forskolin modestly augmented the inhibitory effects of forskolin on tension and [Ca2+]i by methacholine, indicating that the mechanism of desensitization is mediated by the beta-adrenergic receptor/G protein complex. These results indicate that an uncoupling of beta-adrenergic receptor from KCa channels augments Ca2+ mobilization through VDCC and stimulates tachyphylaxis.

Regulation of sodium-iodide-symporter gene expression in human thyrocytes measured by real-time polymerase chain reaction.

The sodium-iodide-symporter (NIS) plays a key role in iodination, the first step in the biosynthesis of the thyroid hormones, and is thought to be critically involved in several thyroid disorders associated with altered iodine up-take. To elucidate the pathogenic role of NIS in these diseases a sensitive technique is needed to measure human NIS gene expression. We established a real time RT-PCR for accurate quantification of hNIS mRNA levels based on fluorescence-labelled hybridisation probes in the LightCycler system. Human NIS expression was investigated in primary cultures of human thyrocytes. After optimisation of PCR conditions less than 10 molecules hNIS were detected with high sensitivity, specificity and reproducibility. Under basal conditions NIS expression varied from 83 to 593 copies per 10 6 GAPDH molecules. Stimulation of thyrocytes with TSH (0.1-10 U/ml) or forskolin (0.1-15 microM) results in a dose- and time-dependent up-regulation of NIS expression reaching a maximum at 10 mU/ml TSH (2211 +/- 761 copies) or 10 microM forskolin (1663 +/- 302 copies) after 24 h. In conclusion, we here established a real-time RT-PCR combining the advantages of rapid thermocycling and online detection of NIS mRNA amplification. The sensitive quantification of human NIS mRNA expression offered by this novel technique may improve analysis of hNIS regulation and measurement of NIS mRNA expression in small biopsies.

Attenuated "cross talk" between kappa-opioid receptors and beta-adrenoceptors in the heart of chronically hypoxic rats.

At 0.1-1 microM, U50488H, a kappa-opioid receptor agonist, inhibited the stimulatory effects of 1 microM isoprenaline, a beta-adrenoceptor agonist, on the electrically induced intracellular Ca2+ ([Ca2+](i)) transient and cAMP accumulation ("cross talk" between kappa-opioid receptors and beta-adrenoceptors) in the presence of 1 microM ICI118,551, a beta(2)-adrenoceptor antagonist in ventricular myocytes from both normoxic and chronically hypoxic rats. Pretreatment with 20 microg/ml cholera toxin increased the ADP-ribosylation of Gsalpha subunits and the electrically induced [Ca2+](i) transient in both normoxic and chronically hypoxic rats. The effects of cholera toxin were inhibited by 1 microM U50488H and the inhibitory effect of U50488H was attenuated in chronically hypoxic rats. Similarly, 1 microM forskolin also increased the electrically induced [Ca2+](i) transient and cAMP accumulation, which were both inhibited by U50488H, in both normoxic and chronically hypoxic rats. The inhibitory effects of 1 microM U50488H were significantly attenuated in chronically hypoxic rats. The results are novel findings, in that the "cross talk" between kappa-opioid receptors and beta-adrenoceptors is attenuated following chronic hypoxia. The attenuation may be due to decreased responses of Gs-protein and adenylyl cyclase to kappa-opioid receptor activation in addition to desensitization of the receptor itself.

Forskolin upregulation of NOS I protein expression in porcine ciliary processes: a new aspect of aqueous humor regulation

To determine whether nitric oxide synthase (NOS) protein expression can be modulated by forskolin in porcine ciliary processes. Western blot analysis using mouse monoclonal antibodies against NOS I (neuronal NOS or nNOS), NOS II (macrophage NOS or macNOS), and NOS III (endothelial NOS or eNOS) was performed in porcine ciliary processes. Expression of NOS I and II was also assessed in the presence or in the absence of 1 microM forskolin exposure (4 and 24 hours). All three NOS isoforms could be detected in isolated porcine ciliary processes. Protein expression for NOS I was about 6 times higher than for NOS III and 15 times higher than for NOS II. In comparison to controls, after forskolin exposure, NOS I protein expression was increased by about 1.5-fold. No change could be observed in NOS II protein expression after forskolin exposure. Nitric oxide is involved in transepithelial fluid transport of different organs (lung, kidney, colon, etc.) and is suspected to play a role in ciliary processes by modulating the aqueous humor production. The present study indicates that forskolin (which increases cAMP production) modulates NOS I protein expression in isolated porcine ciliary processes. These results suggest that selective inhibition of NOS I could be tried for reducing aqueous humor production. The clinical relevance requires further investigations.

Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells.

HCO-dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently, it has been shown that mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO transport have not been reported. Immunoblotting for CFTR showed a single band at approximately 170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect immunofluorescence confocal microscopy indicated that CFTR locates to the apical membrane. Relative changes in apical and basolateral chloride permeability were estimated by measuring the rate of fluorescence quenching of the halide-sensitive indicator 6-methoxy-N-ethylquinolinium iodide during Cl(-) influx in the absence and presence of forskolin (FSK). Apical and basolateral Cl(-) permeability increased 10- and 3-fold, respectively, in the presence of 50 microM FSK. FSK-activated apical chloride permeability was unaffected by H(2)DIDs (250 microM); however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 microM) and glibenclamide (100 microM ) inhibited activated Cl(-) fluxes by 45% and 30%, respectively. FSK-activated basolateral Cl(-) permeability was insensitive to NPPB, glibenclamide, or furosemide but was inhibited 80% by H(2)DIDS. HCO permeability was estimated by measuring changes in intracellular pH in response to quickly lowering bath [HCO]. FSK (50 microM) increased apical HCO permeability by twofold, which was inhibited 42% by NPPB and 65% by glibenclamide. Basolateral HCO permeability was unaffected by FSK. Genistein (50 microM) significantly increased apical HCO and Cl(minus sign) permeability by 1.8- and 16-fold, respectively. When 50 microM genistein was combined with 50 microM FSK, there was no further increase in Cl(-) permeability; however, HCO permeability was reduced to the control level. In summary, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial Cl(-) and HCO transport. Furthermore, there is a cAMP-activated Cl(-) pathway on the basolateral membrane that is not CFTR.

Role of EP(1) and EP(4) PGE(2) subtype receptors in serum-induced 3T6 fibroblast cycle progression and proliferation.

Recent studies have suggested that prostaglandin E(2) (PGE(2)) subtype receptors (EP) are involved in cellular proliferation and tumor development. We studied the role of EP(1) and EP(4) PGE(2) subtype receptor antagonists AH-6809 and AH-23848B, respectively, in serum-induced 3T6 fibroblast proliferation. This was significantly reduced in a dose-dependent manner (IC(50) approximately 100 and approximately 30 microM, respectively) to an almost complete inhibition, without any cytotoxic effect. However, the effect of each antagonist on 3T6 cell cycle progression clearly differed. Whereas the EP(1) antagonist increased the G(0)/G(1) population, the EP(4) antagonist brought about an accumulation of cells in early S phase. These effects were associated with a decrease in cyclin D and E levels in AH-6809-treated 3T6 cells and lower cyclin A levels in AH-23848B-treated fibroblasts with respect to control cells. The G(0)/G(1) accumulation caused by AH-6809 seems to be intracellular Ca(2+) concentration ([Ca(2+)](i)) dependent, because a 6-h 1 microM thapsigargin treatment allowed G(0)/G(1)-arrested cells to enter S phase. Similarly, treatment with 20 microM forskolin for 6 h allowed S-phase and G(2)/M progression of AH-23848B-treated cells. This study shows that the inhibitory effect of the EP(1) and EP(4) antagonists on serum-induced 3T6 fibroblast growth is due to their effect at various levels of the cell cycle machinery, suggesting that PGE(2) interaction with its different subtype receptors regulates progression through the cell cycle by modulating cAMP and [Ca(2+)](i).

CAMP and cGMP do not mediate the vasorelaxation induced by iodinated radiographic contrast media in isolated swine renal arteries.

Vasodilatation is a frequent side effect of radiographic contrast media, partially due to a direct effect on vascular smooth muscles. Our purpose was therefore to examine any possible implication of the cyclic adenosine monophospate (cAMP) and cyclic guanosine monophosphate (cGMP) pathways in contrast media induced vasorelaxation. Isolated segments of swine renal arteries were precontracted with 10 microM phenylephrine and relaxed with iomeprol before and after blockade of the cAMP and cGMP pathways. 80 mM and 160 mM of iomeprol significantly relaxed about 52% and 68% of the precontracted arterial rings, respectively. 10 microM of IBMX, a phosphodiesterase inhibitor, did not increase the relaxant effect of 80 mM iomeprol but increased the relaxations induced by 400 nM forskolin by about 1.9 times (which stimulates the production of cAMP), and by 1 microM sodium nitroprusside (which stimulates the production of cGMP). 1 microM of H89 (an inhibitor of the cAMP-dependent protein kinase), was able to reduce the relaxation induced by 4 microM forskolin by about 2.5 times but had no significant effect on the relaxation induced by 160 mM iomeprol. 10 microM of ODQ (an inhibitor of the soluble guanylate cyclase), could reduce the relaxation induced by 10 microM of SNP but not the one induced by 160 mM iomeprol. Moreover, the absence of endothelial cells did not alter the relaxation induced by iomeprol. The activation of the cAMP and cGMP pathways are not involved in the in vitro relaxation induced by iomeprol in swine renal arteries.

5-HT-receptor-induced changes of the intracellular cAMP level monitored by a hyperpolarization-activated cation channel.

The HvCNG channel from the moth Heliothis virescens is highly sensitive to cAMP concentrations ranging between 0.1 microM and 5 microM. This HvCNG channel was over-expressed in Spodoptera frugiperda (Sf.9) cells to measure endogenous cAMP levels. Hyperpolarization-activated inward currents were measured in the whole-cell patch-clamp configuration with pipettes filled with different cAMP concentrations to calibrate the system. Varying the cAMP concentration between 0 microM and 100 microM in the pipette, the half-maximal activation voltage ( V1/2) was shifted by +28.5+/-1.7 mV. The activation time constant (tau(a)) was used as a parameter for cAMP quantification because it was independent of the expression level of HvCNG channels. tau(a) changed from 1106+/-60 ms at 0 microM cAMP to 265+/-7 ms at a saturating concentration of 1 mM cAMP. A dose-response relationship yielded values of 0.6 microM for the half-maximal cAMP concentration and 1.5 for the Hill coefficient. Activation of endogenous adenylyl cyclases by 50 microM forskolin induced an elevation of the cAMP level by about 1.6+/-0.2 microM. Co-expressions of HvCNG channels in combination with the mouse 5-HT4a- or 5-HT1A- receptors and the corresponding Gs- or Gi-proteins were successful and allowed us to also verify receptor-induced changes of the cAMP level. Stimulation of m5-HT4a-receptors by 0.1 microM 5-HT induced an increase of cAMP of about 4.6+/-1.5 microM, whereas cAMP levels decreased from a control value of 1+/-0.2 microM to 0.41+/-0.1 microM after stimulation of the m5-HT1A-receptors.

Pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal peptide-stimulated cyclic AMP synthesis in rat cerebral cortical slices: interaction with noradrenaline, adrenaline, and forskolin.

Pituitary adenylate cyclase-activating polypeptide (PACAP; 0.001-1 microM) and vasoactive intestinal peptide (VIP; 0.01-1 microM) produced a concentration-dependent stimulation of cyclic AMP (cAMP) formation in rat cerebral cortical slices prelabeled with [3H]adenine. The effects of PACAP38 and PACAP27 were similar, and more efficacious (at 0.1 and 1 microM) than those of VIP. Adrenaline and noradrenaline (each at 100 microM) also stimulated cAMP formation, with the latter compound being more effective. Combination of PACAP38, PACAP27 (each at 0.1 microM) and VIP (1 microM) with adrenaline or noradrenaline resulted in most cases in additive effects, with some supraadditive (PACAP27 plus adrenaline) or subadditive (PACAP38 or VIP plus noradrenaline) fluctuations. In contrast, combination of each of the three peptides with 3 microM forskolin resulted in synergistic effects. These results indicate that in rat cerebral cortex there is no synergism between PACAP or VIP with noradrenaline or adrenaline; however, based on the forskolin data, it seems likely that synergistic effects may take place with VIP or PACAP and other cAMP-stimulating neuroregulators.

Cyclic AMP mediated GSTP1 gene activation in tumor cells involves the interaction of activated CREB-1 with the GSTP1 CRE: a novel mechanism of cellular GSTP1 gene regulation.

The human GSTP1 gene is frequently over-expressed in many human cancers and the expression increases with tumor progression and is associated with a more aggressive biology, poor patient survival, and resistance to therapy. The molecular regulation of the human GSTP1 gene during malignancy is, however, still not well understood. Recently, we reported the presence of a cAMP response element (CRE) in the 5'-region of the human GSTP1 gene, raising the possibility that the cAMP signaling pathway, frequently aberrant in human cancers, may play an important role in the transcriptional activation of the GSTP1 gene in human tumors. In this study, we report that the GSTP1 gene is an early cAMP response gene. Treatment of cells of the human lung carcinoma cell line, Calu-6, with 25 microM forskolin to activate the cAMP pathway resulted in a rapid and significant (sevenfold after 30 min) increase in GSTP1 gene transcripts, which peaked at 12-fold after 4 h. The forskolin-activated GSTP1 transcription in Calu-6 cells was suppressed dose-dependently by a 2-h pre-treatment with 0.1, 1.0, and 10 microM of the adenylate cyclase inhibitor, 2', 5'-dideoxyadenosine. Western blot analysis showed a rapid, fivefold increase, in GSTP1 protein levels after treatment with 25 microM forskolin, with a peak at 2 h post-treatment. The levels of phosphorylated CRE (Ser133) binding protein-1 (CREB-1) increased rapidly, sevenfold at 30 min, and reached 10-fold at 4 h following forskolin treatment. Intracellular cAMP levels also increased rapidly reaching 12-fold at 30 min. Gel mobility shift and supershift assays and DNase/footprinting analyses demonstrated that CREB-1 bZIP and CREB-containing nuclear extracts recognized the GSTP1 CRE with high affinity and specificity. Binding of CREB-1 bZIP to the GSTP1 CRE was abolished when the GSTP1 CRE sequence 5'-CGTCA-3', was mutated at the core nucleotides. Finally, transfection studies using luciferase plasmid constructs showed the GSTP1 CRE to be required for the cAMP-activated gene expression. Together, these findings describe a novel cAMP- and CREB-1-mediated mechanism of transcriptional regulation of the GSTP1 gene and suggest that this may be an important mechanism underlying the increased GSTP1 expression observed in tumors with an aberrant cAMP signaling pathway and in normal cells under conditions of stress, associated with increased intracellular cAMP.

Cyclic AMP increases the survival of ganglion cells in mixed retinal cell cultures in the absence of exogenous neurotrophic molecules, an effect that involves cholinergic activity.

Natural cell death is a well-known degenerative phenomenon occurring during development of the nervous system. The role of trophic molecules produced by target and afferent cells as well as by glial cells has been extensively demonstrated. Literature data demonstrate that cAMP can modulate the survival of neuronal cells. Cultures of mixed retinal cells were treated with forskolin (an activator of the enzyme adenylyl cyclase) for 48 h. The results show that 50 microM forskolin induced a two-fold increase in the survival of retinal ganglion cells (RGCs) in the absence of exogenous trophic factors. This effect was dose dependent and abolished by 1 microM H89 (an inhibitor of protein kinase A), 1.25 microM chelerythrine chloride (an inhibitor of protein kinase C), 50 microM PD 98059 (an inhibitor of MEK), 25 microM Ly 294002 (an inhibitor of phosphatidylinositol-3 kinase), 30 nM brefeldin A (an inhibitor of polypeptide release), and 10 microM genistein or 1 ng/ml herbimycin (inhibitors of tyrosine kinase enzymes). The inhibition of muscarinic receptors by 10 microM atropine or 1 microM telenzepine also blocked the effect of forskolin. When we used 25 microM BAPTA, an intracellular calcium chelator, as well as 20 microM 5-fluoro-2'-deoxyuridine, an inhibitor of cell proliferation, we also abolished the effect. Our results indicate that cAMP plays an important role controlling the survival of RGCs. This effect is directly dependent on M1 receptor activation indicating that cholinergic activity mediates the increase in RGC survival. We propose a model which involves cholinergic amacrine cells and glial cells in the increase of RGC survival elicited by forskolin treatment.