Stimulatory Effect Of Forskolin Research Articles

Difference in expression between AQP1 and AQP5 in porcine endometrium and myometrium in response to steroid hormones, oxytocin, arachidonic acid, forskolin and cAMP during the mid-luteal phase of the estrous cycle and luteolysis.

BackgroundRecently, we demonstrated in vitro that AQP1 and AQP5 in the porcine uterus are regulated by steroid hormones (P4, E2), arachidonic acid (AA), forskolin (FSK) and cAMP during the estrous cycle. However, the potential of the porcine separated uterine tissues, the endometrium and myometrium, to express these AQPs remains unknown. Thus, in this study, the responses of AQP1 and AQP5 to P4, E2 oxytocin (OT), AA, FSK and cAMP in the porcine endometrium and myometrium were examined during the mid-luteal phase of the estrous cycle and luteolysis.MethodsReal-time PCR and western blot analysis.ResultsProgesterone up-regulated the expression of AQP1/AQP5 mRNAs and proteins in the endometrium and myometrium, especially during luteolysis. Similarly, E2 also stimulated the expression of both AQPs, but only in the endometrium. AA led to the upregulation of AQP1/AQP5 in the endometrium during luteolysis. In turn, OT increased the expression of AQP1/AQP5 mRNAs and proteins in the myometrium during mid-luteal phase. Moreover, a stimulatory effect of forskolin and cAMP on the expression of AQP1/AQP5 mRNAs and proteins in the endometrium and myometrium dominated during luteolysis, but during the mid-luteal phase their influence on the expression of these AQPs was differentiated depending on the type of tissue and the incubation duration.ConclusionsThese results seem to indicate that uterine tissues; endometrium and myometrium, exhibit their own AQP expression profiles in response to examined factors. Moreover, the responses of AQP1/AQP5 at mRNA and protein levels to the studied factors in the endometrium and myometrium are more pronounced during luteolysis. This suggests that the above effects of the studied factors are connected with morphological and physiological changes taking place in the pig uterus during the estrous cycle.

Role of nonselective cation channels in spontaneous and protein kinase A-stimulated calcium signaling in pituitary cells

Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity and calcium influx in endocrine pituitary cells, and a role for an unidentified sodium-conducting channel in this process has been proposed. Here we show that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (cAMP-dependent protein kinase) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated Ca(2+) influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 >> TRPC6 > TRPC4 > TRPC5 > TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium.

Co-ordinated action of DISC1, PDE4B and GSK3β in modulation of cAMP signalling

Disrupted-in-Schizophrenia-1 (DISC1) is a risk factor for major mental illness, including schizophrenia and depression.1 DISC1 interacts with multiple proteins involved in intracellular signalling pathways, including two enzymes cAMP phosphodiesterase-4 (PDE4)2 and glycogen synthase kinase-3β (GSK3β),3 that are inhibited by the prototypic antidepressant/antipsychotic Rolipram4 and the mood stabiliser Lithium (LiCl),5 respectively. PDE4 itself has been directly implicated as a risk factor for mental illness,1, 2, 6 whereas the likely involvement of GSK3β in molecular pathways underlying mental illness is long standing and now linked to DISC1.7 DISC1 inhibits GSK3β,3 and we have previously suggested that DISC1 similarly modulates PDE4.2 These enzymes have overlapping binding sites on DISC1,3, 8 suggesting that their function within the DISC1 complex may be related. Moreover, it has recently been demonstrated that GSK3β phosphorylates PDE4D.9 Accordingly, we now provide evidence that pharmacological inhibition of GSK3β decreases PDE4 activity, and demonstrate that the stimulatory effect of Forskolin upon PDE4 activity is modulated by the co-ordinated action of DISC1 and GSK3β.

Direct effects of IL‐15 on corticosterone secretion by rat adrenocortical cells

Cytokines might regulate the function of the hypothalamic-pituitary-adrenal axis. IL-15 is a potent non-T-cell-derived cytokine with IL-2-like activities. It has been shown that IL-15 can reverse the inhibition of glucocorticoids on PBMC. In vitro experiments were designed to assess the direct effect of IL-15 on corticosterone (CORT) secretion in the adrenal zona fasciculata-reticularis (ZFR) cells of male rats. Administration of IL-15 dose dependently decreased the basal and adrenocorticotropin-stimulated release of CORT and production of cAMP in ZFR cells. The stimulatory effect of forskolin (an adenylate cyclase activator) on CORT secretion and accumulation of cAMP in ZFR cells was attenuated by the administration of IL-15. However, 8-Br-cAMP (a cAMP analogue)-stimulated release of CORT was not affected by IL-15. Exogenous administration of IL-15 (10(-7) mol/L) significantly attenuated the pregnenolone (the substrate of 3beta-hydroxysteroid dehydrogenase)- or deoxycorticosterone (the substrate of 11beta-hydroxylase)-induced release of CORT. The results indicate that decrease of CORT secretion by IL-15 is in part because of (i) the decrease of adenylate cyclase activity and cAMP production and (ii) the inhibition of 3beta-hydroxysteroid dehydrogenase and 11beta-hydroxylase activities in rat ZFR cells.

Expression of calcium-sensing receptor in rat colonic epithelium: evidence for modulation of fluid secretion.

The calcium-sensing receptor (CaSR) is activated by extracellular calcium (Ca2+(o)) and mediates many of the known effects of extracellular divalent minerals on body cells. Both surface and crypt cells express CaSR transcripts and protein on both apical and basolateral surfaces. Raising Ca2+(o) elicited increases in intracellular calcium (Ca2+(o)) in both surface and crypt cells with an EC50 of 2 mM. The Ca-induced increase in Ca2+(i) was associated with increases in inositol 1,4,5-trisphosphate and eliminated by U-73129, an inhibitor of phosphatidylinositol-phospholipase C, as well as by thapsigargin. Other CaSR agonists, Gd3+ and neomycin, mimicked these Ca2+(o)-induced responses. Both luminal and bath Ca2+(o), Gd3+, and neomycin induced increases in Ca2+(i) in isolated perfused crypts. The stimulatory effect of forskolin on net fluid secretion in perfused crypts was abolished by increasing Ca2+(o) in either luminal or bath perfusates. Thus both apical and basolateral CaSR on crypt cells are functional and provide pathways modulating net intestinal fluid transport that may have important implications for the prevention and treatment of certain diarrheal diseases associated with elevated cAMP.

Effects of ketoconazole on progesterone and cAMP production in MA-10 mouse Leydig tumor cells.

The effects of ketoconazole (KCZ) on secretion of progesterone and cAMP in Leydig cells were investigated in vitro. MA-10 mouse Leydig tumor cells were used to conduct the experiments. KCZ significantly inhibited the progesterone production from MA-10 cells in a dose dependent fashion between 2 and 20 microM among 1, 2 and 3 h of incubation. There was a statistically significant difference in luteinizing hormone (LH) stimulated progesterone production inhibited by 2 and 20 microm KCZ treatment compared to the control. The effect of KCZ on progesterone biosynthesis in MA-10 cells was mediated by cAMP, since KCZ suppressed basal and LH stimulated cAMP production and content within the same dose range. The stimulatory effects of forskolin and sodium fluoride on the adenylate cyclase system were also inhibited by KCZ. Moreover, dibutyryl cAMP blocked the inhibitory effect on steroidogenesis of KCZ in MA-10 cells. These data indicated that KCZ induced the inhibition of a catalytic component of adenylate cyclase holoenzyme in MA-10 mouse Leydig tumor cells.

Regulation of Tryptophan Hydroxylase Activity in Xenopus laevis Photoreceptor Cells by Cyclic AMP

The aim of this study was to investigate the role of cyclic AMP in the regulation of tryptophan hydroxylase activity localized in retinal photoreceptor cells of Xenopus laevis, where the enzyme plays a key role in circadian melatonin biosynthesis. In photoreceptor-enriched retinas that lack serotonergic neurons, tryptophan hydroxylase activity is markedly stimulated by treatments that increase intracellular levels of cyclic AMP or activate cyclic AMP-dependent protein kinase, including forskolin, phosphodiesterase inhibitors, and cyclic AMP analogues. In contrast, cyclic AMP has no effect on tryptophan hydroxylase mRNA abundance. Experiments using cycloheximide and actinomycin D demonstrate that cyclic AMP exerts its regulatory effect via posttranslational mechanisms mediated by cyclic AMP-dependent protein kinase. The effect of cyclic AMP is independent of the phase of the photoperiod, suggesting that the nucleotide is not a mediator of the circadian rhythm of tryptophan hydroxylase. Cyclic AMP accumulation is higher in darkness than in light, as is tryptophan hydroxylase activity. Furthermore, the stimulatory effect of forskolin and that of darkness are inhibited by H89, an inhibitor of cyclic AMP-dependent protein kinase. In conclusion, cyclic AMP may mediate the acute effects of light and darkness on tryptophan hydroxylase activity of retinal photoreceptor cells.

Effects of evodiamine on the secretion of testosterone in rat testicular interstitial cells.

Evodiamine, a bioactive component isolated from the Chinese medicine Wu-chu-yu, exhibits vasodilative and antianoxic action. Although evodiamine indeed has many biological effects, its effects on the endocrine system are not clear. The present study explored the effects of evodiamine on testosterone secretion in vitro. Rat collagenase-dispersed testicular interstitial cells (TICs) were incubated with evodiamine (0 to 10(-4) mol/L) in the presence or absence of human chorionic gonadotropin (hCG), forskolin, 8-bromo-adenosine 3':5'-cyclic monophosphate (8-Br-cAMP), or steroidogenic precursors (including 25-hydroxycholesterol, pregnenolone, progesterone, 17alpha-hydroxyprogesterone, and androstenedione) at 34 degrees C for 1 hour. The testosterone concentration in the media samples was measured by radioimmunoassay. Evodiamine 10(-4) mol/L was effective to reduce both basal and hCG-stimulated testosterone secretion in rat TICs after 1, 2, or 4 hours of incubation. The stimulatory effect of forskolin on testosterone release in TICs was prevented by administration of evodiamine. Evodiamine 10(-4) mol/L also decreased 8-Br-cAMP- and androstenedione-stimulated testosterone secretion. These results suggest that evodiamine reduces testosterone secretion in rat TICs via a mechanism involving reduced activity of cAMP-related pathways and 17beta-hydroxysteroid dehydrogenase (17beta-HSD).

Dopamine-1 receptor coupling defect in renal proximal tubule cells in hypertension.

The ability of the dopamine-1 (D1)-like receptor to stimulate adenylyl cyclase (AC) and phospholipase C (PLC), inhibit sodium transport in the renal proximal tubule (RPT), and produce natriuresis is attenuated in several rat models of hypertension. Since the inhibitory effect of D1-like receptors on RPT sodium transport is also reduced in some patients with essential hypertension, we measured D1-like receptor coupling to AC and PLC in cultures of human RPT cells from normotensive (NT) and hypertensive (HT) subjects. Basal cAMP concentrations were the same in NT (n=6) and HT (n=4). However, the D1-like receptor agonist fenoldopam increased cAMP production to a greater extent in NT (maximum response=67+/-1%) than in HT (maximum response=17+/-5%), with a potency ratio of 105. Dopamine also increased cAMP production to a greater extent in NT (32+/-3%) than in HT (14+/-3%). The fenoldopam-mediated increase in cAMP production was blocked by SCH23390 (a D1-like receptor antagonist) and by antisense D1 oligonucleotides in both HT and NT, indicating action at the D1 receptor. The stimulatory effects of forskolin and parathyroid hormone-related protein of cAMP accumulation were not statistically different in NT and HT, indicating receptor specificity and an intact G-protein/AC pathway. The fenoldopam-stimulated PLC activity was not impaired in HT, and the primary sequence and expression of the D1 receptor were the same in NT and HT. However, D1 receptor serine phosphorylation in the basal state was greater in HT than in NT and was not responsive to fenoldopam stimulation in HT. These studies demonstrate the expression of D1 receptors in human RPT cells in culture. The uncoupling of the D1 receptor in both rats (previously described) and humans (described here) suggests that this mechanism may be involved in the pathogenesis of hypertension; the uncoupling may be due to ligand-independent phosphorylation of the D1 receptor in hypertension.

Ontogenic Development of the Adenylyl Cyclase Enzyme and the α s, α i1 and α i2 G-protein Regulatory Subunits from Rat Prostate

The expression of α s, α i1 and α i2 G-protein subunits measured by immunoblot increased in the rat prostate during sexual maturation, supporting their involvement in proliferation/differentiation. Northern blotting gave transcripts of 1.8 and 4 kb for α s, 1.4 and 4.5 kb (mainly) for α i1, and 2.4 kb for α i2 with levels suggesting a differential regulation (at transcription or post-transcription for α s, transcription for α i1, and translation for α i2). The stimulatory effects of forskolin, vasoactive intestinal peptide (VIP) and isoproterenol on adenylyl cyclase activity increased between 0.5–3 mo, remained constant up to 12 mo and decreased thereafter, conceivably following the expression of VIP and β-adrenergic receptors. However, G-protein activation of adenylyl cyclase (by GTP and Gpp[NH]p) was maximal at 0.5 mo and then decreased as it occurred with toxin-catalyzed ADP-ribose incorporation to α subunits suggesting that other factors are also involved in the regulation of G-protein activity during rat prostatic development.

An anaesthetic dose of ethanol inhibits cyclic AMP formation by altering the function of GS protein.

The effects of ethanol on cerebral gamma-aminobutyric acid (GABA)B receptor and its related signal transduction systems were studied. [3H]GABA binding to cerebral GABAB receptors in crude synaptic membranes from rat brain was unaffected by ethanol at concentrations up to 100 mM, although 100 mM ethanol suppressed not only the basal but also forskolin-stimulated cAMP formation in cerebral cortical membrane vesicles. The suppressive effect of baclofen and the stimulatory effect of forskolin on cAMP formation was also found to be unaltered by ethanol. Pretreatment of the membrane vesicles with islet-activating protein (IAP), which eliminates the function of Gi protein by its ribosylation, had no effect on the suppressive effects of ethanol on basal and forskolin-stimulated cAMP formation. In contrast, pretreatment of membrane vesicles with 'low pH', which is known to suppress the function of Gs protein, eliminated the suppressive effect of ethanol on cAMP formation. These results suggest that an anaesthetic dose of ethanol, which is below that necessary to cause changes in receptor binding, may not affect the functions of GABAB/Gi, G(O) protein/adenylate cyclase system, but suppresses cAMP formation systems which are coupled with Gs protein.

Modulation of islet G-proteins, α-glucosidehydrolase inhibition and insulin release stimulated by various secretagogues

Guanine nucleotide-binding proteins (G-proteins) are known to act as important modulators of insulin release from the islets of Langerhans. We have recently found that the deoxynojirimycin-derivative emiglitate, a recognized inhibitor of intestinal alpha-glucosidehydrolase activity, is a powerful inhibitor of glucose-induced insulin release. With the use of isolated mouse islets the present investigation was performed in a primary attempt to elucidate whether this inhibitory mechanism in some way was linked to the beta-cell G-protein system. Treatment of freshly isolated islets with pertussis toxin (PTX), which is known to inactivate the G (i)-proteins, abolished the inhibitory effect of the alpha(2)-adrenoceptor agonist clonidine on insulin release stimulated by the phosphodiesterase inhibitor IBMX in the presence of the protein kinase C activator TPA and even changed it into an increase. Emiglitate did not display any inhibitory action on insulin release induced by these secretagogues. Similarly, clonidine-induced inhibition of glucose stimulated insulin release was reversed by PTX. However, PTX did not influence the suppressive action of emiglitate on glucose-induced insulin secretion. In contrast, the adenylate cyclase activator forskolin totally abolished the inhibitory effect of emiglitate, but not that of the glucose analogue mannoheptulose, on glucose-induced insulin release. Moreover, the stimulatory effect of forskolin and cholera toxin (CTX) (activator of G (s)-proteins) on the secretion of insulin was markedly enhanced in the presence of emiglitate. In conclusion, our results suggest that the inhibitory effect of emiglitate on glucose-induced insulin release is not directly related to the G(s)-proteins, but most likely exerted solely through the selective suppression of lysosomal aglucosidehydrolase activity, a step in between the proximal and the distal G(i)-proteins, in glucose induced stimulus-secretion mechanisms. Our data also suggests that the inhibitory action of emiglitate on glucose stimulated insulin release can be compensated for by an increased sensitivity of the cyclic AMP-protein kinase A pathway. Hence, emiglitate might indirectly elicit an increased activity of the G(s)-proteins to facilitate the secretory process.

Role of membrane-permeable ions in renin secretion by renal juxtaglomerular cells

In this study we examine the role of membrane-permeable ions in renin secretion from renal juxtaglomerular (JG) cells. To this end, extracellular Cl- (100 mmol/l) in the culture medium of isolated mouse renal JG cells was replaced by the permeable anion NO3- or by the membrane-impermeable anion isethionate. Alternatively, extracellular Na+ (100 mmol/l) was substituted by the membrane-impermeable cation choline. The effects of these ion substitutions on basal and stimulated renin secretion were then examined. Renin secretion was stimulated by the adenylate cyclase activator forskolin (10 microM), the NO donor sodium nitroprusside (SNP, 100 microM), the calmodulin antagonist calmidazolium (10 microM), by lowering extracellular Ca2+ concentration ([Ca2+]e) with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) (2 mM), and by increasing [Ca2+]e from the normal value of 0.5 to 3 mM. Substitution of extracellular Cl- by isethionate, but not by NO3-, inhibited basal renin release over 20 h of incubation. NO3- also did not change renin secretion stimulated by forskolin, SNP, calmidazolium, EGTA, or by increased [Ca2+]e. Isethionate, on the other hand, markedly attenuated the effects of EGTA and of increased [Ca2+]e, but not the stimulatory effect of forskolin, calmidazolium, or SNP. Substitution of Na+ by choline also attenuated basal renin secretion and renin secretion stimulated by lowering or raising [Ca2+]e. These findings suggest that, with respect to the dependency on permeable ions, at least two different pathways of regulated renin secretion from JG cells exist: a cation- and anion-dependent Ca(2+)-related pathway and a less ion-sensitive pathway for renin secretion activated by adenosine 3',5'-cyclic monophosphate and NO.

Tannin inhibits adenylate cyclase in airway epithelial cells

Tannin, isolated from cotton bracts extract and implicated in the pathogenesis of byssinosis, inhibits adenosine 3',5'-cyclic monophosphate (cAMP) production and Cl- secretion in bovine airway epithelial cells in part by inhibiting adrenergic receptor binding. The purpose of this study was to determine whether tannin affected other parts of the adrenergic-cAMP signal transduction pathway by examining the effect of tannin on guanosine 5'-triphosphate (GTP)-regulatory pathways (G proteins) and on adenylate cyclase activity. cAMP production in confluent airway epithelial cells was measured in the presence of cholera toxin (100 micrograms/ml), an activator of GS proteins, and forskolin (0.1-1,000 microM), a direct activator of adenylate cyclase. Cholera toxin stimulated cAMP production; this response, however, was inhibited in cells pretreated with 50 micrograms/ml tannin. Forskolin (100 microM) stimulated cAMP production 13-fold above baseline values. Tannin pretreatment inhibited the stimulatory effect of forskolin on cAMP release in a dose-dependent manner with a tannin concentration causing 50% inhibition of 7.5 micrograms/ml. The stimulatory effect of forskolin on cAMP release was completely inhibited in cells pretreated with 50 micrograms/ml tannin. The inhibition was reversible 3 h after removal of tannin from the solution. Tannin also inhibited forskolin-stimulated adenylate cyclase activity in a dose-dependent, noncompetitive manner. We conclude that forskolin and cholera toxin stimulate cAMP production in airway epithelial cells and that tannin inhibits the production of cAMP in airway epithelial cells by a direct effect on adenylate cyclase activity.

Characterization of gonadal sex cord-stromal tumor cell lines from inhibin-alpha and p53-deficient mice: the role of activin as an autocrine growth factor.

Inhibin-alpha-deficient mutant mice have been generated by a targeted deletion of the inhibin-alpha gene through homologous recombination in murine embryonic stem cells. Essentially all of the homozygous mutants develop gonadal sex cord-stromal tumors. To investigate their endocrine and proliferative characteristics, gonadal tumor cells were maintained in vitro. Cells from inhibin-alpha-deficient mice multiplied poorly; however, cells from mice deficient in both inhibin-alpha and p53 proliferated rapidly and showed higher saturation density and plating efficiency, thus allowing the establishment of clonal tumor cell lines. Although negligible estrogen and testosterone was produced by the clonal cells, high levels of progesterone were secreted. A clonal testis tumor cell line (inhibin-alpha/p53 deficient) showed no response to exogenous FSH, human CG (hCG), or inhibin A but exhibited a 6- to 8-fold increase in progesterone production in response to forskolin treatment. The stimulatory effect of forskolin was, however, partially blocked by activin treatment. Northern blot analysis revealed inhibin beta A and beta B mRNA expression in these cells. Furthermore, Western blot analyses indicated the secretion of the beta A-subunit protein. We further tested the role of activin on tumor cell growth. Treatment with follistatin, an activin-binding protein, inhibited tumor cell replication in a dose-dependent manner. In contrast, treatment with activin A stimulated tumor cell growth by itself and partially blocked follistatin action. Incorporation of thymidine into DNA of these cells was also stimulated by activin. In addition, treatment with antiactivin A serum inhibited tumor cell replication and blocked the stimulatory action of activin on cell growth. The activin action is likely mediated by specific receptors because cross-linking of [125]activin to the 50-55 kilodalton type I and 75-80 kilodalton type II receptors was found using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Northern blot analysis also revealed follistatin mRNA expression in the tumor cells, suggesting these cells are related to granulosa cells. Our findings indicate that activin can act as an autocrine growth factor in stimulating the proliferation of gonadal tumor cell lines derived from inhibin-alpha and p53-deficient mice and inhibits progesterone production. These tumor cell lines are useful for studies on the regulation of gonadal cell proliferation and steroidogenesis as well as the signaling pathway mediating activin action.

Regulation of tetrahydrobiopterin biosynthesis in cultured dopamine neurons by depolarization and cAMP.

Primary cultures containing embryonic rat brain mesencephalic or hypothalamic dopamine neurons were used to examine the effects of membrane depolarization and elevations of cAMP levels on tetrahydrobiopterin cofactor content. Initial studies showed that 24-h incubations with 8-bromo-cAMP or isobutyl methylxanthine increased cofactor levels in either culture system, whereas the stimulatory effects of forskolin or depolarization of membrane potential were only observed in cultures of hypothalamus. 8-Bromo-cAMP was found to increase cofactor content in a concentration-dependent manner, with increases observed up to 5 mM. The time course of the effect of 8-bromo-cAMP was biphasic. Over the short term, an increase of 50% in cofactor content at 2 and 5 h was detected. Over the long term, by 24-48 h, cofactor levels increased by between 100% and 300%. Studies of cofactor turnover indicated that the long-term increase was due to stimulation of tetrahydrobiopterin biosynthesis with no alteration in degradation rate. Inhibitors of gene transcription and translation prevented the long- but not short-term increase in cofactor content. Levels of GTP cyclohydrolase I mRNA were increased 7-10-fold following 5 h of incubation with 8-bromo-cAMP. Tetrahydrobiopterin biosynthesis within cultured dopamine neurons of the hypothalamus and mesencephalon thus appears to be regulated by a cAMP-dependent mechanism involving enhanced gene expression of enzyme(s) involved in cofactor biosynthesis.

Forskolin stimulates porcine sperm capacitation by increasing calcium uptake

Using the fluorescent calcium indicator fura-2, forskolin was found to dose-dependently cause an immediate increase in the concentration of intracellular free calcium of porcine cauda epididymal sperm. This stimulatory effect of forskolin is due to the enhancement of Ca 2+ uptake by the verapamil-sensitive transporter on the sperm plasma membrane and results in the promotion of the sperm capacitation and subsequent acrosome reaction.

Is forskolin stimulation of rat striatal tyrosine hydroxylase dependent on adenylate cyclase activation?

Because some responses to forskolin are adenosine 3′,5′-cyclic phosphate (cyclic AMP) independent, we investigated the involvement of adenylate cyclase in the stimulatory effect of forskolin on synaptosomal tyrosine hydroxylase (TH) activity of rat striatum. The forskolin analogue, 1,9-dideoxyforskolin, which mimics the cyclic AMP-independent effects of forskolin, was a very weak activator of both striatal adenylate cyclase and tyrosine hydroxylase activities, whereas forskolin stimulated the two enzymes effectively and with similar potencies. Moreover, exposure of synaptosomes to the specific cyclic AMP antagonist Rp-adenosine 3′,5′-cyclic phosphorothioate reduced basal TH activity and counteracted the stimulatory effect on the enzyme activity by submaximal concentrations of forskolin. These results provide circumstantial evidence that in striatal dopaminergic nerve terminals a presynaptic adenylate cyclase mediates the stimulation of TH activity by forskolin.

Altered amounts of G-protein mRNA and cAMP accumulation after long-term opioid receptor stimulation of neurons in primary culture from the rat cerebral cortex

Primary neuronal enriched cultures were incubated with μ (morphine, 10 −5 M), δ (DPDPE, 10 −6 M) and κ (U-50,488H, 10 −5 M) receptor agonists for 5 days, respectively. Thereafter the acute inhibitory actions of μ, δ or κ receptor agonists on forskolin stimulated cAMP accumulation was assayed. The effect of long term opioid treatment on the steady-state level of G-protein mRNA (G αs, G αi-1 and G αi-2) was analyzed using an RNAase protection hybridization assay. Incubation for 5 days with κ receptor agonist resulted in an attenuated ability to decrease the accumulation of cAMP by κ receptors, as well as μ and δ receptors, which was also observed after 5 days of incubation with the δ receptor agonist. Furthermore, the adenylate cyclase responsiveness to forskolin stimulation was markedly reduced in cultures treated with either δ or κ receptor agonists. Five days of incubation with κ receptor agonist resulted in an increase in the levels of G αs and G αi-2 mRNAs. No effects on the amounts of G αs mRNA, G αi-1 mRNA or G αi-2 mRNA were detected after 5 days of δ receptor stimulation. On the other hand, 5 days of μ receptor stimulation decreased the amounts of G αs, G αi-1 and G αi-2 mRNA. Incubation with κ receptor agonist for 24 h resulted in a significant decrease in the forskolin-stimulated accumulation of cAMP. The stimulatory effect of forskolin was further decreased after 3 days incubation with κ receptor agonist. A decreased sensitivity to the acute inhibitory action of μ, δ or κ receptor agonists was seen after 3 days incubation with the κ receptor agonist. The basal accumulation of cAMP was decreased after 24 h and 3 days of incubation with the κ agonist in the neuronal enriched cultures. The present data demonstrate that long-term activation of κ and δ receptors in neuronal enriched cultures from the rat cerebral cortex reduced the stimulated formation of cAMP, induced decreased sensitivity of μ, δ or κ receptors. Furthermore, the present data indicate that long term activation of μ and κ opioid receptors induces alterations in the expression of G-protein mRNA.

Somatostatin inhibition of gastrin gene expression: involvement of pertussis toxin-sensitive and -insensitive pathways

These studies were performed to determine the intracellular pathways involved in regulating gastrin gene expression. The inclusion of 10 −4 M forskolin or 10 −4 M dibutyryl cyclic AMP (DBcAMP) in incubation medium containing dog antral mucosa resulted in 249% and 323% increases, respectively, in gastrin mRNA levels. The stimulatory effects of forskolin and DBcAMP were both inhibited significantly by 10 −6 M somatostatin. Preincubation of antral mucosa with pertussis toxin nearly abolished the inhibitory effects of somatostatin on gastrin mRNA stimulated by forskolin, but had no effect following DBcAMP. To examine whether calcium-dependent pathways might be involved in regulating gastrin gene expression, antral mucosa was incubated with increasing concentrations of calcium or the ionophore ionomycin. Both agents produced only modest increases in gastrin mRNA, which were abolished by the addition of somatostatin to the incubation medium. These studies indicate that somatostatin appears to inhibit gastrin gene expression through mechanisms involving both pertussis toxin-sensitive and -insensitive pathways.