Increase In Cellular cAMP Levels Research Articles

Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

Fibroblast growth factor 21 (FGF21) functions as a polypeptide hormone to regulate glucose and lipid metabolism, and its expression is regulated by cellular metabolic stress. Pyruvate is an important intermediate metabolite that acts as a key hub for cellular fuel metabolism. However, the effect of pyruvate on hepatic FGF21 expression and secretion remains unknown. Herein, we examined the gene expression and protein levels of FGF21 in human hepatoma HepG2 cells and mouse AML12 hepatocytes in vitro, as well as in mice in vivo. In HepG2 and AML12 cells, pyruvate at concentrations above 0.1 mM significantly increased FGF21 expression and secretion. The increase in cellular cAMP levels by adenylyl cyclase activation, phosphodiesterase (PDE) inhibition and 8-Bromo-cAMP administration significantly restrained pyruvate-stimulated FGF21 expression. Pyruvate significantly increased PDE activities, reduced cAMP levels and decreased CREB phosphorylation. The inhibition of exchange protein directed activated by cAMP (Epac) and cAMP response element binding protein (CREB) upregulated FGF21 expression, upon which pyruvate no longer increased FGF21 expression. The increase in plasma pyruvate levels in mice induced by the intraperitoneal injection of pyruvate significantly increased FGF21 gene expression and PDE activity with a reduction in cAMP levels and CREB phosphorylation in the mouse liver compared with the control. In conclusion, pyruvate activates PDEs to reduce cAMP and then inhibits the cAMP–Epac–CREB signaling pathway to upregulate FGF21 expression in hepatocytes.

The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway.

Inhibitory effects of benzodiazepines on the adenosine A2B receptor mediated secretion of interleukin-8 in human mast cells

The activation of adenosine A2B receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A2B receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3–3μM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A2B receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1μM. In addition, diazepam, 4′-chlorodiazepam and flunitrazepam (1–30μM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10μM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10μM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A2B receptors, excluding a direct action of benzodiazepines on human adenosine A2B receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A2B receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

Red wine acts through a familiar drug target

Resveratrol, a natural polyphenol first identified and named by a Japanese chemist in 1940 [1], is present in many plant products, including grape skin. It has been the focus of much attention recently because it provides a variety of potential health benefits, in particular protection from various age-related conditions (such as diabetes mellitus, vascular diseases, cognitive disorders, and cancer), and increases life span [2–4]. Given that red wine is rich in resveratrol, and that moderate wine consumption has been shown to lower cardiovascular risk [5], the intake of resveratrol through the drinking of red wine may contribute to the ‘‘French paradox’’—which refers to the fact that the mortality due to cardiovascular diseases is lower in France than in other European countries despite similar lifestylerelated risk factors [6]. Sirt1, an NAD-dependent protein deacetylase, is thought to play an important role in the action of resveratrol [2]. Resveratrol stimulates the activity of Sirt1, and overexpression of Sirt1 protects mice from age-related diseases [2]. Moreover, orthologs of Sirt1 appear to contribute to life span extension by resveratrol in worms and flies [2]. Whereas an early study suggested that resveratrol directly stimulates the activity of Sirt1 [7], this notion has been challenged by more recent studies [8, 9]. Resveratrol was found to stimulate the activity of AMPactivated protein kinase (AMPK) [10], which can increase cellular NAD levels and thereby stimulate the activity of Sirt1 [11], but the effect of resveratrol on AMPK is also likely not direct. The direct target of resveratrol for its health-promoting effects has thus remained a mystery. Park et al. [12] have now revealed that a group of enzymes that serve as common drug targets are also targeted by resveratrol. The cyclic nucleotides cAMP and cGMP are both important messenger molecules in intracellular signaling. An increase in the cellular levels of cAMP and cGMP is achieved through their synthesis by adenylyl cyclase and guanylyl cyclase, respectively. Conversely, the levels of these molecules are reduced through their hydrolysis by phosphodiesterases (PDEs). Mammalian PDEs constitute a large family of enzymes that are categorized into 11 subtypes (PDE1 to PDE11) encoded by 21 different genes [13]. Individual PDEs differ in their affinity for substrates, tissue distribution, and mode of regulation, and together they share a broad range of biological tasks [13]. Various pharmacological inhibitors of PDEs have been developed, some of which are marketed as drugs for a wide variety of conditions, including bronchial asthma (theophylline), peripheral artery disease (cilostazol), heart failure (amrinone), cognitive disorders (vinpocetine), depression (rolipram), and erectile dysfunction (sildenafil, vardenafil, and tadalafil) [13]. Park et al. [12] have now shown that resveratrol inhibits the activity of several PDEs (PDE1, -3, and -4, but not -2 or -5) in vitro by directly competing with cAMP to bind to these isozymes (Fig. 1). Inhibition of PDEs results in an increase in cellular cAMP levels and the consequent activation of two signaling pathways: the cAMP-dependent protein kinase (PKA) pathway and the Epac1 (exchange protein directly activated by cAMP 1) pathway [14]. Park et al. [12] found that both Sirt1 and AMPK were activated by resveratrol in a manner dependent on Epac1, consistent with the notion that the cellular accumulation of cAMP, likely due to PDE inhibition, is the primary cellular event triggered by resveratrol. Calciumand calmodulinW. Ogawa (&) T. Hosooka Division of Diabetes and Endocrinology, Department of Internal Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan e-mail: ogawa@med.kobe-u.ac.jp

Involvement of prostaglandin E2 in production of beta-amyloid peptides both in vitro and in vivo

Amyloid-β peptides (Aβ, generated by proteolysis of the β-amyloid precursor protein (APP) by β- and γ-secretases, play an important role in the pathogenesis of Alzheimer's disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we examined the effect of prostaglandin E2 (PGE2), a strong inducer of inflammation, on the production of Aβ in vitro. We also examined the role of PGE2 in pathogenesis of AD in vivo. The production of Aβ in cells expressing a mutant type of APP (APPsw) was examined by ELISA. Cognitive performance was assessed by a hidden-platform Morris-type water maze. PGE2 stimulated production of Aβ and, of the four main subtypes of PGE2 receptors (EP1-4), EP2 and EP4 receptors are responsible for this PGE2-stimulated production of Aβ. Activation of EP2 and EP4 receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP2 receptor-mediated but not EP4 receptor-mediated stimulation of the production of Aβ by PGE2. In contrast, inhibitors of endocytosis suppressed EP4 receptor-mediated but not EP2 receptor-mediated stimulation. PGE2-dependent internalization of EP4 receptor was observed and cells expressing a mutant EP4 receptor lacking the internalization activity did not exhibit PGE2-stimulated production of Aβ. A surface biotinylation assay revealed that the γ-secretase internalizes in a manner dependent on both PGE2 and EP4 receptors. On the other hand, in vivo, transgenic mice expressing APPsw showed lower levels of Aβ in the brain and less apparent cognitive deficit, when they were crossed with mice lacking EP4 receptors. Administration of antagonist for EP4 receptor also improved cognitive deficit of transgenic mice expressing APPsw. These results suggest that PGE2-stimulated production of Aβ involves EP4 receptor-mediated endocytosis of γ-secretase, and EP2 receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of AD. Results also suggest that PGE2-mediated activation of EP4 receptor is involved in the production of Aβ in vivo and in the pathogenesis of AD and that antagonists for EP4 receptor are therapeutically beneficial for AD.

Adrenergic Regulation of the HERG Potassium Channel Biosynthesis and Function

The HERG (human ether-a-go-go related gene) potassium channel is linked to the hereditary Long QT Syndrome (LQTS, locus LQT2) and is a drug binding target in the acquired LQTS. HERG channels are regulated by several intracellular signaling pathways that together contribute to the overall modulation of the cardiac potassium current IKr in normal and disease states. Previous studies have established the acute regulation of HERG current though the beta-adrenergic pathway with an increase in cellular cAMP levels, activation of protein kinase A (PKA) and direct phosphorylation of the HERG channel. Regulation by the alpha-adrenergic system involving protein kinase C (PKC) activity has been less well characterized. Chronic effects of adrenergic stimulation on the HERG channel have not been studied. We have found that 24-hour stimulation with increased intracellular cAMP levels or phorbol esters result in distinct increases in HERG protein abundance. This increase in protein levels is not transcriptionally mediated as shown by qRT-PCR and corresonds more to increased production rather than reduced degradation of channel protein. We are currently investigating the underlying mechanism of this kinase-responsive enhancement of steady-state HERG protein levels. We have found that PKA activity can be co-precipitated with HERG, as they exist in a complex. We are using a cell-free in-vitro translation system to isolate and determine the contribution of signaling components such as PKA and PKC during HERG synthesis at the ER. We have found that addition of ATP and purified PKA together accelerates generation of new HERG protein, indicating a direct regulation of translation rate. Ongoing studies using this system will allow us to dissect the molecular mechanisms that regulate HERG channel synthesis.

Prostaglandin E2 Stimulates the Production of Amyloid-β Peptides through Internalization of the EP4 Receptor

Amyloid-beta (Abeta) peptides, generated by the proteolysis of beta-amyloid precursor protein by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease. Inflammation is also important. We recently reported that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta through EP(2) and EP(4) receptors, and here we have examined the molecular mechanism. Activation of EP(2) and EP(4) receptors is coupled to an increase in cellular cAMP levels and activation of protein kinase A (PKA). We found that inhibitors of adenylate cyclase and PKA suppress EP(2), but not EP(4), receptor-mediated stimulation of the Abeta production. In contrast, inhibitors of endocytosis suppressed EP(4), but not EP(2), receptor-mediated stimulation. Activation of gamma-secretase was observed with the activation of EP(4) receptors but not EP(2) receptors. PGE(2)-dependent internalization of the EP(4) receptor was observed, and cells expressing a mutant EP(4) receptor lacking the internalization activity did not exhibit PGE(2)-stimulated production of Abeta. A physical interaction between the EP(4) receptor and PS-1, a catalytic subunit of gamma-secretases, was revealed by immunoprecipitation assays. PGE(2)-induced internalization of PS-1 and co-localization of EP(4), PS-1, and Rab7 (a marker of late endosomes and lysosomes) was observed. Co-localization of PS-1 and Rab7 was also observed in the brain of wild-type mice but not of EP(4) receptor null mice. These results suggest that PGE(2)-stimulated production of Abeta involves EP(4) receptor-mediated endocytosis of PS-1 followed by activation of the gamma-secretase, as well as EP(2) receptor-dependent activation of adenylate cyclase and PKA, both of which are important in the inflammation-mediated progression of Alzheimer disease.

Activation of CREB by PKA promotes the chondrogeneic differentiation of chick limb bud mesenchymal cells

Cyclic AMP‐mediated signaling pathways regulate a number of cellular functions. In this study, we examined the regulatory role of cAMP signaling pathways in chondrogenesis of chick limb bud mesenchymal cells in vitro. Forskolin, which increases cellular cAMP levels by the activation of adenylate cyclase, enhanced chondrogenic differentiation. Inhibition of PKA with specific inhibitors (H89 or KT5720) blocked pre‐cartilage condensation stage, indicating that chondrogenesis is regulated by the increase in cellular cAMP level and subsequent activation of PKA. Downstream signaling pathway of PKA leading to gene expression was investigated by examination of several nuclear transcription factors. Forskolin treatment increased transcription level for a cartilage‐specific marker gene Sox9. However, inhibition of PKA with H89 led to restore expression of Sox9, indicating PKA activity was required to regulate the expression of Sox9 in chondrogenesis. In addition, CREB was highly phosphorylated at early stage of mesenchyme culture, and followed by progressive dephosphorylation. CBP and ATF, another CRE related proteins were transiently expressed at the early stage of chondrogenesis with a pattern similar to CREB phosphorylation. Electrophoretic mobility shift assays confirmed that the binding activity of CREB to the CRE is closely correlated to the phosphorylation pattern of CREB. Therefore, cAMP‐mediated signal transduction to nuclear events for the induction of genes appeared to be required at the early stage of chick limb bud chondrogenesis.

Phosphodiesterase 7A inhibitor ASB16165 suppresses proliferation and cytokine production of NKT cells

A possible involvement of phosphodiesterase 7A (PDE7A) in proliferation and function of NKT cells was examined using ASB16165, a selective inhibitor for PDE7A. Stimulation of isolated murine NKT cells with anti-CD3 antibody plus IL-2 induced not only cell proliferation but production of cytokines including IFN-γ, TNF-α, IL-17 and IL-22. ASB16165 significantly inhibited the CD3/IL-2-stimulated cell proliferation and production of all the cytokines examined. Forskolin (an activator of adenylyl cyclase) and dibutyryl cAMP also exerted inhibitory effects on the cell proliferation and cytokine production of NKT cells. In addition, Rp-8-Br-cAMPS, an inhibitor of protein kinase A (PKA), reversed the suppressive effects of ASB16165 against NKT cells. These results suggest that PKA/cAMP as well as PDE7A is involved in regulation of cell proliferation and cytokine production of NKT cells, and that the inhibitory effects of ASB16165 in NKT cells shown here are mediated by increase in cellular cAMP level. Our findings also raise the possibility that PDE7A inhibitor including ASB16165 may be useful for treatment of the diseases in which NKT cells have pathogenic roles.

Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor Potential Vanilloid 1 Sensitization

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the mu opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the mu opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the mu opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for fine-tuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

Pentoxifylline impedes migration in B16F10 melanoma by modulating Rho GTPase activity and actin organisation

Cancer cell migration is a hallmark of metastatic cascade and compounds that can intervene in this process are clinically important. Pentoxifylline (PTX), a methyl xanthine derivative, inhibits B16F10 melanoma lung homing by inhibiting F10 invasion, MMP secretion and adhesion to matrix components. However, its effect on B16F10 migration remained unexamined, which we investigated in the present study. PTX significantly inhibits F10 migration in scratch wound assay. Elevation in cAMP levels inhibits F10 migration and PTX mediated inhibition of the process was found to be, in part, due to an increase in cellular cAMP levels. PTX induces Protein Kinase A (PKA) activity and PKA inhibitor partly reversed its effects on F10 motility. RhoA and Rac1 GTPases induce B16F10 motility and PTX was found to inhibit migration by affecting these molecules. Stress fibres and lamellipodial protrusions reduced significantly. This was accompanied with inhibition in RhoA and Rac1 membrane localisation. A stark inhibition in RhoA-GTP bound form was also observed. Taken together, the results indicate that PTX, through its phosphodiesterase action, inhibits RhoGTPases and associated actin organisation in B16F10 melanoma, thereby inhibiting cell motility.

Involvement of Prostaglandin E2 in Production of Amyloid-β Peptides Both in Vitro and in Vivo

Amyloid-beta peptides (Abeta), generated by proteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases, play an important role in the pathogenesis of Alzheimer disease (AD). Inflammation is also believed to be integral to the pathogenesis of AD. Here we show that prostaglandin E(2) (PGE(2)), a strong inducer of inflammation, stimulates the production of Abeta in cultured human embryonic kidney (HEK) 293 or human neuroblastoma (SH-SY5Y) cells, both of which express a mutant type of APP. We have demonstrated using subtype-specific agonists that, of the four main subtypes of PGE(2) receptors (EP(1-4)), EP(4) receptors alone or EP(2) and EP(4) receptors together are responsible for this PGE(2)-stimulated production of Abeta in HEK293 or SH-SY5Y cells, respectively. An EP(4) receptor antagonist suppressed the PGE(2)-stimulated production of Abeta in HEK293 cells. This stimulation was accompanied by an increase in cellular cAMP levels, and an analogue of cAMP stimulated the production of Abeta, demonstrating that increases in the cellular level of cAMP are responsible for the PGE(2)-stimulated production of Abeta. Immunoblotting experiments and direct measurement of gamma-secretase activity suggested that PGE(2)-stimulated production of Abeta is mediated by activation ofgamma-secretase but not of beta-secretase. Transgenic mice expressing the mutant type of APP showed lower levels of Abeta in the brain, when they were crossed with mice lacking either EP(2) or EP(4) receptors, suggesting that PGE(2)-mediated activation of EP(2) and EP(4) receptors is involved in the production of Abeta in vivo and in the pathogenesis of AD.

The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is a human pathogenic fungus that switches from a saprobic mycelium to a pathogenic yeast. Consistent with the morphological transition being regulated by the cAMP-signalling pathway, there is an increase in cellular cAMP levels both transiently at the onset (< 24 h) and progressively in the later stages (> 120 h) of the transition to the yeast form, and this transition can be modulated by exogenous cAMP. We have cloned the cyr1 gene encoding adenylate cyclase (AC) and established that its transcript levels correlate with cAMP levels. In addition, we have cloned the genes encoding three Gα (Gpa1–3), Gβ (Gpb1) and Gγ (Gpg1) G proteins. Gpa1 and Gpb1 interact with one another and the N-terminus of AC, but neither Gpa2 nor Gpa3 interacted with Gpb1 or AC. The interaction of Gpa1 with Gpb1 was blocked by GTP, but its interaction with AC was independent of bound nucleotide. The transcript levels for gpa1, gpb1 and gpg1 were similar in mycelium, but there was a transient excess of gpb1 during the transition, and an excess of gpa1 in yeast. We have interpreted our findings in terms of a novel signalling mechanism in which the activity of AC is differentially modulated by Gpa1 and Gpb1 to maintain the signal over the 10 days needed for the morphological switch.

PVD9902, a porcine vas deferens epithelial cell line that exhibits neurotransmitter-stimulated anion secretion and expresses numerous HCO3− transporters

Epithelial ion transport disorders, including cystic fibrosis, adversely affect male reproductive function by nonobstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. In the present study, porcine vas deferens epithelial cells were isolated by standard techniques, and the cells spontaneously immortalized to form a porcine vas deferens epithelial cell line that we have titled PVD9902. Cells were maintained in continuous culture for >4 yr and 200 passages in a typical growth medium. Frozen stocks were generated, and thawed cells exhibited growth characteristics indistinguishable from their nonfrozen counterparts. Molecular and immunocytochemical studies confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grew as electrically tight (>6,000 ohms x cm2), confluent monolayers that responded to forskolin with an increase in short-circuit current (I(sc); 8 +/- 1 microA/cm2) that required Cl-, HCO3(-), and Na+, and was partially sensitive to bumetanide. mRNA was expressed for a number of anion transporters, including CFTR, electrogenic Na+-HCO3(-) cotransporter 1b (NBCe1b), downregulated in adenoma, pendrin, and Cl-/formate exchanger. Both forskolin and isoproterenol caused an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers responded to physiological (i.e., adenosine, norepinephrine) and pharmacological [i.e., 5'-(N-ethylcarboxamido)adenosine, isoproterenol] agonists with increases in I(sc). Unlike their freshly isolated counterparts, however, PVD9902 cells did not respond to glucocorticoid exposure with an increase in amiloride-sensitive I(sc). RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as mRNA for the alpha- and gamma-subunits of the epithelia Na+ channels (alpha- and gamma-ENaC), but not beta-ENaC. Nonetheless, PVD9902 cells recapitulated most observations in freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function.

Characterization of the Gαs Regulator Cysteine String Protein

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca2+ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP1–198 (full-length), CSP1–112, and CSP1–82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with Gαs and increases steady-state GTP hydrolysis. CSP1–198 modulation of Gαs was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP1–112 was Hsc70-SGT-independent. CSP1–112 preferentially associated with the inactive GDP-bound conformation of Gαs. Consistent with the stimulation of GTP hydrolysis, CSP1–112 increased guanine nucleotide exchange of Gαs. The interaction of native Gαs and CSP was confirmed by coimmunoprecipitation and showed that Gαs associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gαs and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.

Luteinizing Hormone Receptors Are Self-Associated in Slowly Diffusing Complexes during Receptor Desensitization

We have previously shown that rat LH receptors (LHRs) occupied by human CG (hCG) exhibit slow receptor lateral diffusion and are self-associated. Here we have examined whether LHRs become self-associated and enter slowly diffusing structures in response to hormone binding and whether these receptors retain this organization while in the desensitized state. Before hormone exposure, wild-type rat LHRs coupled at the C terminus to enhanced green fluorescent protein (GFP-LHR-wt) exhibited fast lateral diffusion, as assessed by fluorescent photobleaching recovery (FPR) methods, and most receptors were laterally mobile. After 30 min exposure to hCG and subsequent removal of hormone by low pH wash, hormone challenge at any time within the next 4 h produced no increase in cellular cAMP levels. During this time, LHRs were either laterally immobile or exhibited slower lateral diffusion. When LHRs were again responsive to binding of hormone, the rate of receptor lateral diffusion had become significantly faster and the fraction of mobile receptors was again large. Desensitized LHRs were also self-associated and present in microscopically visible clusters on the plasma membrane. Fluorescence energy transfer (FET) methods were used to measure the extent of interaction between receptors coupled to either GFP or to yellow fluorescent protein (YFP). Before hormone treatment, there was essentially no energy transfer between LHRs. After desensitization of the receptors by 30 min exposure to hCG, energy transfer efficiency increased to 18%. Values for FET efficiency between desensitized receptors decreased over time, and receptors were responsive to hormone only after measurable energy transfer had completely disappeared. Together these results suggest that desensitized LHRs exist in large, slowly diffusing structures containing self-associated receptors and that these structures must dissipate before the receptor can again respond to hormone.

Luteinizing Hormone Receptors Are Self-Associated in Slowly Diffusing Complexes during Receptor Desensitization

We have previously shown that rat LH receptors (LHRs) occupied by human CG (hCG) exhibit slow receptor lateral diffusion and are self-associated. Here we have examined whether LHRs become self-associated and enter slowly diffusing structures in response to hormone binding and whether these receptors retain this organization while in the desensitized state. Before hormone exposure, wild-type rat LHRs coupled at the C terminus to enhanced green fluorescent protein (GFP-LHR-wt) exhibited fast lateral diffusion, as assessed by fluorescent photobleaching recovery (FPR) methods, and most receptors were laterally mobile. After 30 min exposure to hCG and subsequent removal of hormone by low pH wash, hormone challenge at any time within the next 4 h produced no increase in cellular cAMP levels. During this time, LHRs were either laterally immobile or exhibited slower lateral diffusion. When LHRs were again responsive to binding of hormone, the rate of receptor lateral diffusion had become significantly faster and the fraction of mobile receptors was again large. Desensitized LHRs were also self-associated and present in microscopically visible clusters on the plasma membrane. Fluorescence energy transfer (FET) methods were used to measure the extent of interaction between receptors coupled to either GFP or to yellow fluorescent protein (YFP). Before hormone treatment, there was essentially no energy transfer between LHRs. After desensitization of the receptors by 30 min exposure to hCG, energy transfer efficiency increased to 18%. Values for FET efficiency between desensitized receptors decreased over time, and receptors were responsive to hormone only after measurable energy transfer had completely disappeared. Together these results suggest that desensitized LHRs exist in large, slowly diffusing structures containing self-associated receptors and that these structures must dissipate before the receptor can again respond to hormone.

The Escherichia coli Heat Labile Toxin Binds to Golgi Membranes and Alters Golgi and Cell Morphologies Using ADP-ribosylation Factor-dependent Processes

The fate of the catalytic subunit of the Escherichia coli heat labile toxin (LTA(1)) was studied after expression in mammalian cells to assess the requirement for ADP-ribosylation factor (ARF) binding to localization and toxicity and ability to compete with endogenous ARF effectors. A progression in LTA(1) localization from cytosol to binding Golgi stacks to condensation of Golgi membranes was found to correlate with the time and level of LTA(1) expression. At the highest levels of LTA(1) expression the staining of LTA and both extrinsic and lumenal Golgi markers all became diffuse, in a fashion reminiscent of the actions of brefeldin A. Thus, LTA(1) binds to the Golgi and can alter its morphology in two distinct ways. However, point mutants of LTA(1) that are defective in the ability to bind activated ARF were also unable to bind Golgi membranes or modify Golgi morphology. Co-expression of mutants of ARF3 that regained binding to these same mutant LTA(1) proteins restored the localization and activities of the toxin. Thus, binding to ARF is required both for the localization of the toxin to the Golgi and for effects on Golgi membranes. A correlation was also seen between the ability of LTA mutants to bind ARF and the increase in cellular cAMP levels. These results demonstrate the importance of ARF binding to the toxicity and cellular effects of the ADP-ribosylating bacterial toxin and reveal that mutants defective in binding ARF retain basal ADP-ribosylation activity but are the least toxic LTA(1) mutants yet described, making them the best candidates for development as mucosal adjuvants.

Increased Glomerular Cell Heparan Sulfates in vitro by Ciclosporin A: A Possible Explanation of Its Beneficial Effect in Idiopathic Nephrotic Syndrome

Background/Aim: In idiopathic nephrotic syndrome (INS), ciclosporin A (CsA) was shown to decrease proteinuria, an effect explained by its immunologic and hemodynamic actions. In order to determine whether CsA could have a direct action on glomerular cells, we studied the effect of CsA on glomerular cells in vitro, particularly on glycosaminoglcycans (GAG) and heparan sulfates (HS) which are decreased in INS patients. Methods: Human glomerular epithelial cells and rat mesangial cells were cultured at various concentrations of CsA. HS were quantified using a cationic membrane after metabolic labeling. Results: Mesangial cell GAG and HS and epithelial cell HS increased significantly when cells were cultured with CsA. For both cell types this increase was prevailing on the secreted fraction of HS in comparison with the cellular fraction. CsA induced also an increase in cellular cAMP levels, but the effect of CsA was not transduced via a cAMP pathway. Conclusions: CsA is able to increase glomerular GAG and HS in vitro. As this effect of CsA was the opposite effect on glomerular cells to the effect of plasma from INS patients, we conclude that this direct action of CsA on glomerular cells could explain in part the effect of this drug in decreasing proteinuria in INS.

Pituitary adenylate cyclase-activating polypeptide acts synergistically with relaxin in modulating ovarian cell function in rats.

The interactive effects of pituitary adenylate cyclase-activating polypeptide (PACAP) and relaxin on the secretion of gelatinases, involved in matrix remodeling, in ovarian theca-interstitial cells and granulosa cells, were investigated in gonadotropin-primed immature rats. The gelatinases secreted from cultured cells were analyzed using gelatin zymography and scanning densitometry. We have previously shown that relaxin stimulated the secretion of a 71 kDa gelatinase, identified as a type IV collagenase (matrix metalloproteinase 2), in rat theca-interstitial cells. This study has demonstrated that PACAP27 and PACAP38, with similar potency, dose-dependently enhanced relaxin-induced secretion of 71 kDa gelatinase, whereas PACAP alone had no effect. In rat granulosa cells, both PACAP27 and PACAP38 alone dose-dependently increased the secretion of a 63 kDa gelatinase. In addition, this study has shown that cAMP signaling pathway mediators act similarly to that of PACAP on gelatinase secretion in rat ovarian cells. Cholera toxin, forskolin and 8-bromoadenosine cAMP augmented relaxin-induced secretion of 71 kDa gelatinase in theca-interstitial cells, and alone they had no effect. These mediators also increased the secretion of 63 kDa gelatinase in granulosa cells. It is well known that the increase in cellular cAMP level is associated with the morphological rounding-up phenomenon in granulosa cells. This study has shown that PACAP and cAMP pathway mediators, but not relaxin, could cause such changes in cell shape in granulosa cells as well as in theca-interstitial cells. In conclusion, this study provides original findings that PACAP acts synergistically with relaxin in stimulating the secretion of gelatinases in rat ovarian theca-interstitial cells and granulosa cells. This supports the idea that relaxin and PACAP may serve as ovarian physiological mediators of gonadotropin function in facilitating the ovulatory process. In addition, PACAP appears to act through the cAMP signaling pathway to affect biological functions in ovarian cells, whereas relaxin does not.