A novel adenylyl cyclase type 5 inhibitor that reduces myocardial infarct size even when administered after coronary artery reperfusion.

We developed a novel adenylyl cyclase type 5 (AC5) inhibitor, C90, that reduces myocardial infarct size even when administered after coronary reperfusion. This is key, since it is not practical to administer a drug to a patient with myocardial infarction before revascularization, and is one reason why so many prior drugs, which reduced infarct in experimental animals, failed in clinical trials. C90 is the most potent AC5 inhibitor, as exhibited by its IC50 value for AC5 inhibition, which was 5 times lower than the next most potent AC5 inhibitor. C90 reduced cAMP in response to forskolin in wild type mice by 42%, but no longer reduced cAMP in response to forskolin in mice with disruption of AC5, indicating that the mechanism of C90 was specific for AC5 inhibition. Compared with vehicle treatment, C90 reduced infarct size by 64% at a dose of 0.6 mg/kg. Thus, C90 is a novel, selective and potent AC5 inhibitor that reduces infarct size, when delivered after coronary artery reperfusion, rendering it potentially clinically useful. It also reduces beta-adrenergic receptor signaling, which will provide additional benefit to patients with coronary artery disease or heart failure.

Escitalopram Ameliorates Tau Hyperphosphorylation and Spatial Memory Deficits Induced by Protein Kinase A Activation in Sprague Dawley Rats.

Here, we investigated the effect of escitalopram pretreatment on protein kinase A (PKA)-induced tau hyperphosphorylation and spatial memory deficits in rats using western blot and behavioral tests, respectively. We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. We measured the total and activity-dependent Ser9-phosphorylated levels of glycogen synthase kinase (GSK)-3ß in hippocampal extracts. No significant change in the total level of GSK-3ß was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3ß. We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). Furthermore, we identified other important kinases and phosphatases, such as protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2, and MAP kinase kinase-1/2, that have previously been reported to play a crucial role in tau phosphorylation; however, we did not detect any significant change in the activation of these kinases or phosphatases in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin. These data provide the first evidence that escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3ß signaling pathway.

Forskolin: genotoxicity assessment in Allium cepa.

Forskolin, a diterpene, 7ß-acetoxy-8,13-epoxy-1α,6ß,9α-trihydroxy-labd-14-en-11-one (C22H34O7) isolated from Coleus forskohlii, exerts multiple physiological effects by stimulating the enzyme adenylate cyclase and increasing cyclic adenosine monophosphate (cAMP) concentrations. Forskolin is used in the treatment of hypertension, congestive heart failure, eczema, and other diseases. A cytogenetic assay was performed in Allium cepa to assess possible genotoxic effects of forskolin. Forskolin was tested at concentrations 5-100 µM for exposure periods of 24 or 48 h. Treated samples showed significant reductions in mitotic index (p < 0.05) and increases in the frequency of chromosome aberrations (p < 0.01) at both exposure times. The treated meristems showed chromosome aberrations including sticky metaphases, sticky anaphases, laggard, anaphase bridges, micronuclei, polyploidy, fragments, breaks, and C-mitosis. Forskolin may cause genotoxic effects and further toxicological evaluations should be conducted to ensure its safety.

More than just hormones: H295R cells as predictors of reproductive toxicity.

Many of the commonly observed reproductive toxicities associated with therapeutic compounds can be traced to a disruption of the steroidogenic pathway. We sought to develop an in vitro assay that would predict reproductive toxicity and be high throughput in nature. H295R cells, previously validated as having an intact and functional steroidogenic pathway, were treated with 83 known-positive and 79 known-negative proprietary and public-domain compounds. The assay measured the expression of the key enzymes STAR, 3ßHSD2, CYP17A1, CYP11B2, CYP19A1, CYP21A2, and CYP11A1 and the hormones DHEA, progesterone, testosterone, and cortisol. We found that a Random Forest model yielded a receiver operating characteristic area under the curve (ROC AUC) of 0.845, with sensitivity of 0.724 and specificity of 0.758 for predicting in vivo reproductive toxicity with this in vitro assay system.

Dietary Coleus forskohlii extract generates dose-related hepatotoxicity in mice.

Coleus forskohlii root extract (CFE) represented by its bioactive constituent 'forskolin' is popularly used as a natural weight-lowering product, but the association of its use with liver-related risks is very limited. In the present study, the effect of standardized CFE with 10% forskolin on liver function of mice was examined. Mice were given 0-5% CFE in an AIN93G-based diet for 3-5 weeks. Food intake, body weights, relative organ weights and liver marker enzymes [aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP)] combined with histophatological analysis were assessed. CFE (0-0.5%) only had minimal effects on food intake and body weight whereas a significant difference was observed in mice receiving the highest dose (5% CFE). The extract 0.05-5% dose-dependently decreased visceral fat weight by between 16% and 63%, and a dose-dependent several folds increase was observed in liver weights and plasma AST, ALT and ALP activities with quick onset apparent after only 1 week of 0.5% CFE intake. The hepatic effect persisted throughout the 3-weeks course but was restored towards normalization within 1 week after withdrawal of treatment. Liver histology of mice fed 0.5% CFE for 3 weeks showed hepatocyte hypertrophy and fat deposition. In contrast, none of the hepatic responses measured were altered when mice were given a diet containing pure forskolin alone at the dose corresponding to its content in 0.5% CFE. The present study clearly indicated that forskolin was not involved in the CFE-induced hepatotoxicity and was caused by other unidentified constituents in CFE which warrants further studies.

Immune-regulatory transcriptional responses in multiple organs of Atlantic salmon after tributyltin exposure, alone or in combination with forskolin.

Tributyltin (TBT) is a widespread marine pollutant that influences physiological conditions of fish and other aquatic organisms. In addition to effects on reproduction, the immune system has been proposed as a possible target for TBT effects. In the present study, the effects of TBT exposure were examined on the expression of genes involved in immune system compentence in liver and head kidney of Atlantic salmon, in the presence and absence of a second-messenger activator (forskolin). Juvenile salmon were force-fed a diet containing TBT (0-solvent control, 0.1, 1, or 10 mg/kg fish) for 72 h. Consequently, fish from the control group and 10-mg/kg TBT group were exposed to the adenylate cyclase (AC) activator forskolin (200 µg/L) for 2 or 4 h. Forskolin was selected for this study because it is known to exhibit potent immune system enhancement by activating macrophages and lymphocytes. After sacrifice, liver and head kidney were sampled and transcript changes for interleukin (IL)-1ß, IL-10, transforming growth factor (TGF) ß, interferon (INF) α, INFγ, tumor necrosis factor (TNF) α, Mx3, and insulin-like growth factor (IGF)-1 were determined in both tissues by quantitative polymerase chain reaction (qPCR) using gene-specific primers. TBT, when given alone and also in combination with forskolin, decreased IL-1ß, TNFα, IFNγ, IFNα, Mx3, and IGF-1 gene expression. In contrast, IL-10 and TGFß transcripts were increased after TBT exposure alone and also in combination with forskolin. Generally, these effects were largely dependent on TBT dose and time of exposure when given in combination with forskolin. Overall, our findings suggest a possible immunomodulatory effect of TBT, possibly involving cAMP activation.

Curcumin inhibits renal cyst formation and enlargement in vitro by regulating intracellular signaling pathways.

Autosomal dominant polycystic kidney disease, a common inherited disease affecting about 1/1000 and 1/400 live births, is characterized by massive enlargement of fluid-filled cysts and eventually causes renal failure. The purpose of this study is to identify the inhibitory effect of curcumin on renal cyst development and to investigate the inhibitory mechanism. Madin-Darby canine kidney (MDCK) cyst model and murine embryonic kidney cyst model were used to evaluate inhibitory activity. Cell viability, proliferation, apoptosis, CFTR function and expression, and signaling pathways in MDCK cells were determined to explore the mechanism of cyst inhibition. Curcumin was found to significantly inhibit MDCK cyst development. At maximum dose curcumin caused 62% inhibition of the cyst formation (IC(50) was 0.12 µM). Curcumin slowed cyst enlargement in both MDCK cyst model and embryonic kidney cyst model with dose-response relationship. Curcumin neither induced cytotoxicity nor apoptosis in MDCK cells at <100 µM. Curcumin failed to affect the chloride transporter CFTR expression and function. Interestingly, curcumin inhibited forskolin-promoted cell proliferation and promoted the tubule formation in MDCK cells, which indicates curcumin promotes MDCK cell differentiation. Furthermore, curcumin reduced the intracellular signaling proteins Ras, B-raf, p-MEK, p-ERK, c-fos, Egr-1, but increased Raf-1 and NAB2 in MDCK cells exposed to forskolin. These results define that curcumin inhibits renal cyst formation and enlargement and suggest that curcumin might be developed as a candidate drug for polycystic kidney disease.

Mechanisms of L-DOPA-induced cytotoxicity in rat adrenal pheochromocytoma cells: implication of oxidative stress-related kinases and cyclic AMP.

L-DOPA therapy for Parkinson's disease has a double-edge effect on nigrostriatal dopaminergic neurons: L-DOPA increases the intracellular level of dopamine, but it induces neuron cytotoxicity in a concentration-dependent manner. To investigate the molecular signaling mechanisms that underlie the concentration-dependent effects of L-DOPA on cell viability, the activities of mitogen-activated protein kinases (MAPKs) and apoptotic enzymes were measured in rat adrenal pheochromocytoma (PC12) cells in the presence of a low concentration (20 muM) and high concentrations (100-200 muM) of L-DOPA. At the low concentration, L-DOPA was not cytotoxic and its presence increased the activities of extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, BadSer112, Bcl-2, and caspase-12. At the high concentrations, L-DOPA was cytotoxic and stimulated the activities of ERK1/2, p38 MAPK, c-Jun N-terminal kinase (JNK)1/2, BadSer155, caspase-12 and caspase-3. The increased levels of ERK1/2 and BadSer155 in the presence of high concentrations of L-DOPA did not protect against L-DOPA-mediated cytotoxicity. In addition, the levels of L-type Ca(2+) channel-sensitive intracellular cyclic AMP (cAMP) and Ca(2+) were elevated in the presence of L-DOPA, and the increase in the levels of intracellular cAMP may also play a role in cellular viability, since cAMP levels and cytotoxicity increased in parallel with L-DOPA concentrations and the addition of forskolin in the medium increased cytotoxicity in a concentration-dependent manner. These results suggest that, at a low and non-toxic concentration, L-DOPA may promote cell survival by increasing the activities of ERK1/2, BadSer112 and Bcl-2, while, at high concentrations, L-DOPA activates the caspase-3 cell death enzyme through the JNK1/2 and p38 MAPK signaling pathways as well as endoplasmic reticulum stress that activates caspase-12. Intracellular cAMP levels may also play a role here. The results may lead to an effective therapy for Parkinson's disease.

Modulation of acute steroidogenesis, peroxisome proliferator-activated receptors and CYP3A/PXR in salmon interrenal tissues by tributyltin and the second messenger activator, forskolin.

There are uncertainties regarding the role of sex steroids in sexual development and reproduction of gastropods, leading to the recent doubts as to whether organotin compounds do inhibit steroidogenic enzymes in these species. These doubts have led us to suspect that organotin compounds may affect other target molecules, particularly signal transduction molecules or secondary mediators of steroid hormone and lipid synthesis/metabolism. Therefore, we have studied the effects of TBT exposure through food on acute steroidogenesis, PPARs and CYP3A responses in the presence and absence of a cyclic AMP (cAMP) activator, forskolin. Two experiments were performed. Firstly, juvenile salmon were force-fed once with diet containing TBT doses (0.1, 1 and 10mg/kg fish) dissolved in ethanol and sampled after 72h. Secondly, fish exposed to solvent control and 10mg/kg TBT for 72h were transferred to new tanks and exposed to waterborne forskolin (200microg/L) for 2 and 4h. Our data show that juvenile salmon force-fed TBT showed modulations of multiple biological responses in interrenal tissues that include, steroidogenesis (cAMP/PKA activities; StAR and P450scc mRNA, and plasma cortisol), and mRNA for peroxisome proliferator-activated receptor (PPAR) isoforms (alpha, beta, gamma), acyl-CoA oxidase-1 (ACOX1) and CYP3A/PXR (pregnan X receptor). In addition, forskolin produced differential effects on these responses both singly and also in combination with TBT. Overall, combined forskolin and TBT exposure produced higher effects compared with TBT exposure alone, for most of the responses (cortisol, PPARbeta, ACOX1 and CYP3A). Interestingly, forskolin produced PPAR isoform-specific effects when given singly or in combination with TBT. Several TBT mediated toxicity in fish that includes thymus reduction, decrease in numbers of lymphocytes, inhibition of gonad development and masculinization, including the imposex phenomenon have been reported. When these effects are considered with the present findings, it suggests that studies on mechanisms of action or field studies may reveal endocrine, reproductive or other effects of TBT at lower concentrations than those reported to date from subchronic tests of fishes. Since the metabolic fate of organotin compounds may contribute to the toxicity of these chemicals, the present findings may represent some new aspects of TBT toxicity not previously reported.

Modulation of steroidogenesis and estrogen signalling in the estuarine killifish (Fundulus heteroclitus) exposed to ethinylestradiol.

Previous studies have shown that mummichog (Fundulus heteroclitus; a lunar, asynchronous-spawning killifish of the western Atlantic) exposed to 17alpha-ethynylestradiol (EE2) exhibit decreased plasma reproductive steroid levels, decreased gonadal steroid production, increased plasma vitellogenin, decreased fecundity and impaired fertilization. The objective of this study was to determine the potential mechanisms by which EE2 depresses gonadal steroidogenesis and influences estrogen signalling in the mummichog. Adult recrudesced fish were exposed to the potent synthetic estrogen, ethinylestradiol (EE2; 0-270ng/L) for 14 days. Following exposure, gonadal tissue was removed and incubated for 24h with stimulators of steroidogenesis, including forskolin; 25-OH cholesterol; or pregnenolone. Testosterone production was decreased in basal, forskolin-stimulated and pregnenolone-stimulated EE2-exposed males, indicating effects on the steroidogenic pathway both at and downstream of cholesterol mobilization to P450 side-chain cleavage (P450scc) and/or P450scc conversion of cholesterol to pregnenolone. Hepatic transcript levels of estrogen receptor alpha (ERalpha) and vitellogenin were increased in EE2-treated males compared to control recrudescing males and females confirming an estrogenic response. Hepatic heat shock protein 90 (Hsp90), a chaperoning molecule involved in estrogen signalling, was not affected by EE2 exposure at either the transcript or protein level. However, higher levels of Hsp90 observed in the membrane fractions of female fish raise interesting questions regarding the influence of gender on Hsp90's role in estrogen signalling. These results demonstrate that EE2 can alter steroid production at specific sites within the steroidogenic pathway and can stimulate hepatic estrogen signalling, providing important information regarding the molecular mechanisms underlying the endocrine response of the mummichog to exogenous estrogen.

Molecular changes associated with teratogen-induced cyclopia.

BACKGROUND: Exposure of zebrafish embryos to a number of teratogens results in cyclopia, but little is known about the underlying molecular changes. METHODS: Using zebrafish embryos, we compare the effects cyclopamine, forskolin, and ethanol delivered starting just before gastrulation, on gene expression in early axial tissues and forebrain development. RESULTS: Although all three teratogens suppress gli1 expression, they do so with variable kinetics, suggesting that while suppression of Shh signaling is a common outcome of these three teratogens, it is not a common cause of the cyclopia. Instead, all teratogens studied produce a series of changes in the expression of gsc and six3b present in early axial development, as well as a later suppression of neural crest cell marker dlx3b. Ethanol and forskolin, but not cyclopamine, exposure reduced anterior markers, which most likely contributes to the cyclopic phenotype. CONCLUSIONS: These data suggest that each teratogen exposure leads to a unique set of molecular changes that underlie the single phenotype of cyclopia.

Chronic hyperalgesic priming in the rat involves a novel interaction between cAMP and PKCepsilon second messenger pathways.

Toward the goal of defining new pharmacological targets for the treatment of chronic pain conditions, in previous studies we established a model, termed 'hyperalgesic priming,' in which an acute inflammatory stimulus causes a long-lasting latent susceptibility to hyperalgesia induced by subsequent exposures to the inflammatory mediator, prostaglandin E2 (PGE2). Those investigations suggested the hypothesis that priming induces a novel linkage between the PGE2-activated second messenger cascade and the epsilon isoform of protein kinase C (PKCepsilon). In the present study, comparison of dose-response relations for hyperalgesia produced by PGE2, forskolin, 8-Br-cAMP, or the protein kinase A (PKA) catalytic subunit, in primed versus normal animals, demonstrated that priming-induced enhancement of the PGE2-activated second messenger cascade occurs downstream to adenylate cyclase and upstream to PKA. Therefore, PGE2-induced hyperalgesia in the primed animal is enhanced by the recruitment of a novel cAMP/PKCepsilon signaling pathway in addition to the usual cAMP/PKA pathway. These observations suggest that pharmacological disruption of the novel interaction between cAMP and PKCepsilon might provide a route toward the development of highly specific methods to reverse cellular processes that underlie chronic pain states.

Empirical bayes gene screening tool for time-course or dose-response microarray data.

An efficient method to reduce the dimensionality of microarray gene expression data from thousands or tens of thousands of cDNA clones down to a subset of the most differentially expressed cDNA clones is essential in order to simplify the massive amount of data generated from microarray experiments. An extension to the methods of Efron et al. [Efron, B., Tibshirani, R., Storey, J., Tusher, V. (2001). Empirical Bayes analysis of a microarray experiment. J. Am. Statist. Assoc. 96:1151-1160] is applied to a differential time-course experiment to determine a subset of cDNAs that have the largest probability of being differentially expressed with respect to treatment conditions across a set of unequally spaced time points. The proposed extension, which is advocated to be a screening tool, allows for inference across a continuous variable in addition to incorporating a more complex experimental design and allowing for multiple design replications. With the current data the focus is on a time-course experiment; however, the proposed methods can easily be implemented on a dose-response experiment, or any other microarray experiment that contains a continuous variable of interest. The proposed empirical Bayes gene-screening tool is compared with the Efron et al. (2001) method in addition to an adjusted model-based t-value using a time-course data set where the toxicological effect of a specific mixture of chemicals is being studied.

Complex shape changes in isolated rat osteoclasts: involvement of protein kinase C in the response to calcitonin.

The cytoplasmic spreading of osteoclasts has been used to assess responsiveness to agents such as calcitonin and associated signal transduction mechanisms. Although cyclic AMP and intracellular calcium are known mediators of calcitonin effects in osteoclasts, the role of protein kinase C (PKC) is less clear. We have used time-lapse videomicroscopy of isolated rat osteoclasts to characterize shape changes induced by calcitonin, forskolin, and phorbol 12-myristate-13-acetate (PMA) in the absence and presence of PKC blockers. Treatment with calcitonin reduced cytoplasmic plan area but increased perimeter length, resulting in a characteristic "stellate" appearance, whereas forskolin produced "nonstellate" contraction. The response of osteoclasts to PMA was dose dependent. High concentrations (10(-7)-10(-6) M) produced biphasic responses with transitory, calcitonin-like "stellate" contraction followed by sustained expansion, whereas low concentrations (10(-11)-10(-9) M) produced expansion only. The effects of low-concentration PMA could be prevented by pretreatment with a PKC blocker, whereas the effects of high concentrations were only partially inhibited. The effects of forskolin were unchanged by pretreatment with the PKC blocker. Treatment with calcitonin in the presence of various PKC blockers resulted in paradoxical transient expansion followed by contraction. These results indicate that calcitonin-induced shape change in osteoclasts is a complex process involving protein kinase C in addition to cyclic AMP-dependent mechanisms and possibly other factors.

Down-regulation of protein kinase C by parathyroid hormone and mezerein differentially modulates cAMP production and phosphate transport in opossum kidney cells.

We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na(+)-dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production, Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH-, cholera toxin (CTX)-, and forskolin (FSK)-stimulated cAMP production, suggesting enhanced Gs alpha coupling and increased adenylyl cyclase activity. However, PKA- and PKC-dependent regulation of Na/Pi were blocked in MEZ-treated cells. The PTH-induced decrease in cAMP production was associated with a reduction in membrane-associated PKC activity while MEZ-induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down-regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist-specific activation and/or down-regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.

Regulation of signal transduction and DNA fragmentation in thymocytes by ethanol.

We demonstrated previously that ethanol enhances apoptosis of murine thymocytes. In this report, we determined intracellular cAMP and cytosolic free calcium ([Ca2+]i) levels in mouse thymocytes following acute exposure to ethanol and investigated the involvement of cAMP, [Ca2+]i, protein kinase A (PKA), and protein kinase C (PKC) in thymocyte apoptotic death induced by ethanol. It was found that ethanol did not alter basal cAMP levels, but produced a dose-dependent, prolonged small [Ca2+]i increase within thymocytes. This dose dependence of [Ca2+]i increase was paralleled by the magnitude of DNA fragmentation induced by ethanol at various concentrations. Additionally, the ethanol-enhanced DNA fragmentation was blocked by H7, a PKC inhibitor, but not by potent PKA inhibitors having little or no effect on PKC. These data suggest that both [Ca2+]i increase and PKC activation triggered by ethanol may belong to the signal pathway(s) leading to thymocyte programmed death.

The effect of forskolin on the teratogenicity of methylxanthines in the chick embryo heart.

Interactions between forskolin and methylxanthines, including caffeine and isobutylmethylxanthine (IBMX), in the developing chick embryo heart were investigated. Forskolin, a potent activator of adenylate cyclase, was administered to young chick embryos (Hamburger-Hamilton stage 24) together with caffeine or IBMX at doses where each agent alone caused minimal embryotoxicity. The incidence of malformation in the embryonic chick heart or aorta induced by caffeine (5 x 10(-7) or 5 x 10(-6) mol) and IBMX (1 or 2.5 x 10(-6) mol) significantly increased with coadministration of forskolin (1 x 10(-9) mol). Cardiovascular malformations included ventricular septal defect, double outlet right ventricle, and aortic arch anomalies. These results indicate that forskolin potentiates the teratogenicity of caffeine or IBMX on the cardiovascular system in the chick embryo and suggest that this potentiation may be related to increase intracellular cAMP due to stimulation of adenylate cyclase (forskolin) and inhibition of phosphodiesterase (methylxanthines).

An in vitro system to screen for diarrheagenic chemicals.

We examined an in vitro system to screen for diarrheagenic chemicals using an established intestinal cell line (T84 human colonic carcinoma). The cells were grown on Millicell-PCF (polycarbonate membrane) wells. The cells were seeded at approximately 5 x 10(6) cells/30mm well and incubated for 9-11 days in a 5% CO2 incubator saturated with water at 37 degrees C. The culture medium was a 1:1 mixture of Ham's F12 and Dulbecco's MEM with 5% fetal bovine serum and 25 micrograms/ml gentamicin sulfate. The well containing cells was removed from the incubator and mounted in a modified Ussing chamber for measurement of short-circuit current (ISC). Chemical-induced increases in ISC are usually indicative of electrogenic epithelial Cl- secretion, which is associated with diarrheagenic effects in animals and humans. T84 cells grown on Millicell-PCF membrane responded with an increase in ISC after basolateral addition of the cholinergic (muscarinic) agonist carbachol, prostaglandin E2, 16,16-dimethylprostaglandin E2, and forskolin, while non-diarrheagenic prostaglandin D2 did not affect ISC. Based on our results, this in vitro system has the potential to be adapted as a rapid screen for detecting diarrheagenic chemicals.

Further confirmation of the role of adenyl cyclase and of cAMP-dependent protein kinase in primary afferent hyperalgesia.

Recent evidence has suggested that cAMP plays a role as a second messenger in the decrease in nociceptive threshold (or hyperalgesia) produced by agents acting on primary afferent terminals. In support of this hypothesis we report that intradermal injection of a direct activator of adenyl cyclase, forskolin, produces a dose-dependent hyperalgesia in the rat. The duration of this hyperalgesia was prolonged by the phosphodiesterase inhibitors, isobutylmethylxanthine and rolipram. Forskolin hyperalgesia was antagonized by the Rp isomer of cyclic adenosine-3'5'-monophosphothioate, an analog of cAMP that prevents the phosphorylation of the cAMP protein kinase. The Rp isomer of cyclic adenosine-3'5'-monophosphothioate also inhibited the hyperalgesia induced by a membrane-permeable analogue of cAMP, 8-bromocyclic adenosine monophosphate, as well as the hyperalgesia induced by agents that are presumed to act directly on primary afferent nociceptors: prostaglandin E2, prostaglandin I2, (8R,15S)-dihydroxyicosa(5E-9,11,13Z)tetraenoic acid; and the adenosine A2-agonist 2-phenylaminoadenosine. Although the cAMP second messenger system contributes to primary afferent hyperalgesia, we found no evidence for a contribution of protein kinase C. Thus, hyperalgesia induced by prostaglandin E2, prostacyclin (prostaglandin I2), (8R,15S)-dihydroxyicosa(5E-9,11,13Z)tetraenoic acid, the adenosine A2-agonist 2-phenylaminoadenosine, 8-bromocyclic adenosine monophosphate and the direct activator of adenyl cyclase, forskolin, were not significantly attenuated by the selective inhibition of protein kinase C by the 19-31 fragment of protein kinase C. Two other inhibitors of protein kinase C, sphingosine and staurosporine, also failed to attenuate prostaglandin E2-induced hyperalgesia.