cAMP Antagonist Rp-cAMP Research Articles

Suppression of Rat Retinal Ganglion Cell Death by PACAP Following Transient Ischemia Induced by High Intraocular Pressure

Glaucoma is a neurodegenerative disease in which increasing intraocular pressure leads to the progressive loss of retinal ganglion cells (RGCs) and blindness. Here, we report a neuroprotective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) against RGC loss induced by high intraocular pressure in the rat. Vehicle or PACAP (1 fM to 1,000 pM) solution was injected into the vitreous body once after induction of a high intraocular pressure (110 mmHg). Seven days later, the number of viable RGCs was reduced to 45% of that in the intact control. However, PACAP treatment significantly reduced this RGC death in a bimodal manner, with peaks at 10 fM and 10-100 pM. The cAMP antagonist Rp-cAMP significantly blocked the neuroprotective effect of PACAP at both high and low doses, whereas the MAP kinase inhibitor PD-98059 only prevented the effect of the low dose of PACAP. These findings suggest that PACAP has bimodal effects in the neuroprotection of RGCs against ischemia and that these effects are mediated via different signaling pathways.

Growth Hormone Secretagogues Reduce Transient Outward K+ Current via Phospholipase C/Protein Kinase C Signaling Pathway in Rat Ventricular Myocytes

Endogenous ghrelin and its synthetic counterpart hexarelin are peptide GH secretagogues (GHS) that exert a positive ionotropic effect in the cardiovascular system. The mechanism by which GHS modulate cardiac electrophysiology properties to alter myocyte contraction is poorly understood. In the present study, we examined whether GHS regulates the transient outward potassium current (I(to)) as well as the putative intracellular signaling cascade responsible for such regulation. GHS and experimental agents were applied locally onto freshly isolated adult Sprague-Dawley rat ventricular myocytes and action potential morphology and I(to) was recorded using nystatin-perforated whole-cell patch-clamp recording technique. Under current clamp, ghrelin and hexarelin (10 nm) significantly prolonged action potential duration. Under voltage clamp, hexarelin and ghrelin inhibited I(to) in a concentration-dependent manner. This inhibition was abolished in the presence of the GHS receptor (GHS-R) antagonist [D-Lys(3)]GH-releasing peptide-6 (10 microm) and GHS-R1a-specific antagonist BIM28163 (1 microm). GHS-induced I(to) inhibition was totally reversed by the phospholipase C inhibitor U73122 (5 microm) and protein kinase C inhibitors GO6983 (1 microm) and calphostin C (0.1 microm) but not by the cAMP antagonist Rp-cAMP (100 microm) or the PKA inhibitor H89 (1 microm). We conclude that hexarelin and ghrelin activate phospholipase C and protein kinase C signaling cascade through the stimulation of the GHS-R, resulting in a decrease in the I(to) current and subsequent prolongation of action potential duration.

Norepinephrine induced calcitonin secretion in rat medullary thyroid carcinoma 6-23 cells: interaction between intracellular calcium and cAMP.

Catecholamines are known to stimulate calcitonin secretion in C-cells by a receptor mediated pathway, but details regarding the postreceptor events are unknown. Since norepinephrine (NE) influences intracellular calcium concentration [Ca2+]i and cAMP levels in C-cells, we used different adrenergic agonists and antagonists to investigate the effect of NE on [Ca2+]i and cAMP accumulation, and on calcitonin secretion in rat MTC-6-23 cells. NE stimulated intracellular cAMP accumulation and calcitonin secretion dose-dependent, with 10(-7) mol/l causing maximal stimulation. The NE induced increase in cAMP accumulation/calcitonin secretion could be decreased to baseline by equimolar amounts of the beta-adrenergic blocker propanolol. The alpha-blocker phentolamine did not significantly influence NE stimulated calcitonin secretion even at high concentrations. The beta-adrenergic agonist fenoterol proved to be as effective as NE in stimulating cAMP accumulation/calcitonin secretion. Activation of inhibitory G-proteins by the adenosine A1 receptor analogue N6-phenylisopropyladenosine at 10(-6) mol/l completely blocked NE stimulated calcitonin secretion. NE stimulated calcitonin secretion was also completely blocked by the cAMP antagonist RpcAMPs. The calcium channel blocker verapamil significantly inhibited NE stimulated calcitonin secretion, but interestingly increased NE stimulated cAMP accumulation. We conclude that NE induced calcitonin secretion is mediated through beta-receptors coupled to adenylate cyclase via G-proteins. cAMP and changes in [Ca2+]i are necessary for NE induced calcitonin secretion. There seems to be a complex interaction between the two pathways even regarding events occurring distal to cell membrane.

Endothelial factors mediate aldosterone release via PKA-independent pathways

Aldosterone synthesis is primarily regulated by angiotensin II and potassium ions. In addition, endothelial cell-secreted factors have been shown to regulate mineralocorticoid release. We analyzed the pathways that mediate endothelial cell-factor-induced aldosterone release from adrenocortical cells, NCI-H295R using endothelial cell-conditioned medium (ECM). The cAMP antagonist Rp-cAMP caused a 44% decrease in the ECM-induced aldosterone release but inhibition of cAMP-dependent PKA had no effect on aldosterone release. Interestingly, inhibition of cAMP-regulated guanine nucleotide exchange factor Epac with brefeldin-A decreased the ECM-induced aldosterone release by 45%. Similarly, inhibition of p38 MAP-kinase; PI-3-kinase and PKB significantly reduced the ECM-induced aldosterone release whereas inhibition of ERK1/2 and PKC did not decrease aldosterone release. These results provide evidence for the existence of a cAMP-dependent but PKA-independent pathway in mediating the ECM-induced aldosterone release and the significant influence of more than one signaling mechanism.

Involvement of Tetrodotoxin-Resistant Na+ Current and Protein Kinase C in the Action of Growth Hormone (GH)-Releasing Hormone on Primary Cultured Somatotropes from GH-Green Fluorescent Protein Transgenic Mice

GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca2+ concentration and GH secretion. Na+ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca2+ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na+ current that is mediated by cAMP. TTX-resistant (TTX-R) Na+ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na+ channels and a TTX-R Na+ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100 nm) increased the TTX-R Na+ current in a reversible manner. The GHRH-induced increase in TTX-R Na+ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na+ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, Gö-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na+ current, but GHRH had no further effect on the current. Na+-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na+ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na+ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.

Gliotoxin from Aspergillus fumigatus affects phagocytosis and the organization of the actin cytoskeleton by distinct signalling pathways in human neutrophils

Gliotoxin is a mycotoxin having a considerable number of immuno-suppressive actions and is produced by several moulds such as Aspergillus fumigatus. In this study, we investigated its toxic effects on human neutrophils at concentrations corresponding to those found in the blood of patients with invasive aspergillosis. Incubation of the cells for 10 min with 30–100 ng/ml of gliotoxin inhibited phagocytosis of either zymosan or serum-opsonized zymosan without affecting superoxide production or the exocytosis of specific and azurophil granules. Gliotoxin also induced a significant re-organization of the actin cytoskeleton which collapsed around the nucleus leading to cell shrinkage and the disappearance of filopodia. This gliotoxin-induced actin phenotype was reversed by the cAMP antagonist Rp-cAMP and mimicked by pCPT-cAMP indicating that it probably resulted from the deregulation of intracellular cAMP homeostasis as previously described for gliotoxin-induced apoptosis. By contrast, gliotoxin-induced inhibition of phagocytosis was not reversed by Rp-cAMP but by arachidonic acid, another member of a known signalling pathway affected by the toxin. This suggests that gliotoxin can affect circulating neutrophils and favour the dissemination of A. fumigatus by inhibiting phagocytosis and the consequent killing of conidia.

A Model of the Roles of Essential Kinases in the Induction and Expression of Late Long-Term Potentiation

The induction of late long-term potentiation (L-LTP) involves complex interactions among second-messenger cascades. To gain insights into these interactions, a mathematical model was developed for L-LTP induction in the CA1 region of the hippocampus. The differential equation-based model represents actions of protein kinase A (PKA), MAP kinase (MAPK), and CaM kinase II (CAMKII) in the vicinity of the synapse, and activation of transcription by CaM kinase IV (CAMKIV) and MAPK. L-LTP is represented by increases in a synaptic weight. Simulations suggest that steep, supralinear stimulus-response relationships between stimuli (e.g., elevations in [Ca 2+]) and kinase activation are essential for translating brief stimuli into long-lasting gene activation and synaptic weight increases. Convergence of multiple kinase activities to induce L-LTP helps to generate a threshold whereby the amount of L-LTP varies steeply with the number of brief (tetanic) electrical stimuli. The model simulates tetanic, θ-burst, pairing-induced, and chemical L-LTP, as well as L-LTP due to synaptic tagging. The model also simulates inhibition of L-LTP by inhibition of MAPK, CAMKII, PKA, or CAMKIV. The model predicts results of experiments to delineate mechanisms underlying L-LTP induction and expression. For example, the cAMP antagonist RpcAMPs, which inhibits L-LTP induction, is predicted to inhibit ERK activation. The model also appears useful to clarify similarities and differences between hippocampal L-LTP and long-term synaptic strengthening in other systems.

CAMP-PKA signaling pathway regulates bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts

Cathepsin K (Cat K) is the major cysteine protease expressed in osteoclast and is thought to play a key role in matrix degradation during bone resorption. It is shown that the intracellular maturation of Cat K was prevented by the cAMP antagonist, Rp-cAMP, and the protein kinase A (PKA) inhibitors of KT5720 and H89. In contrast, forskolin, an adenylate cyclase agonist, rather induced Cat K processing and maturation in osteoclast. Furthermore, to determine whether Cat K processing and maturation signaling involves protein kinase C (PKC), mouse total bone cells were treated with calphostin C, a specific inhibitor of PKC, however, no effect was observed, indicating that PKC calphostin C did not affect to osteoclast-mediated Cat K processing and maturation in osteoclast. Thus, it is indicated that the cAMP-PKA signaling pathway regulate Cat K maturation in osteoclast. Since secreted proenzymes have the potential to reenter the cell via M6P receptor, to prevent this possibility, we tested cAMP antagonist Rp-cAMP and the PKA inhibitors KT5720 and H89 in the absence or presence of M6P. Inhibition of Cat K processing by Rp-cAMP, KT5720 or H89 was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of Rp-cAMP, KT5720 and H89, which dose-dependently inhibited in vitro bone resorption with potency similar to that observed for inhibition of Cat K processing.

Early changes in prostaglandins precede bone formation in a rabbit model of heterotopic ossification

We have tested the hypothesis that the formation of heterotopic ossification (HO) in a rabbit model is correlated with a local increase in specific prostaglandins that may modulate mechanisms of ossification. Rabbits were sacrificed at 1 to 21 days following the daily forcible flexion of immobilized knees. The extraction and analysis of prostaglandins (PG) E2, F2alpha, D2, 6-keto-F1alpha, and thromboxane B2 in vastus intermedius muscles of manipulated legs revealed increases compared to control hindlimbs for all five prostaglandins, albeit of differing magnitude. The earliest increase was observed for PGF2alpha after 24 h (to 2.6-fold of control) with peak levels observed at day ten (185-fold of control). PGE2 was increased above control from 2 to 21 days following manipulation, with a peak level of 33-fold of control after 10 days. In a separate arm of the study, the role of PGE2 was investigated through the use of pharmacological antagonist of the PGE2 receptors and one of its second messengers, cAMP. Rabbits were preadministered the PGE2/PGD receptor antagonist AH 6809 or the cAMP antagonist Rp-cAMP prior to undergoing the regimen of limb immobilization and passive exercise. Both AH 6809 and Rp-cAMP were found to prevent the later development of radiographically documented heterotopic ossification in 15 out of 16 animals, thus identifying prostaglandins as being required for the development of ectopic bone. In this latter group, all but one pharmacologically treated animal showed an absence of HO at 3, 4, 5, or 6 weeks. These findings suggest an obligate cascade of prostaglandins for HO that offers the potential for novel prophylactic therapies, including those that target receptors for specific prostaglandins.

The effect of prostaglandin E 1 on ion currents of NG108-15 cells

The aim of this study was to elucidate the mechanism by which prostaglandin E 1 (PGE 1) acts on ion currents of whole-cell voltage-clamped NG108-15 neuroblastoma × glioma hybrid cells. Ruptured and perforated patch were used. The holding current at −70 mV, the current–voltage curve produced by ramp pulses from −70 to 0 mV and the T-type and hva (high-voltage-activated) Ca 2+ currents associated with rectangular pulses were recorded. Bath application of PGE 1 (0.2 or 3 μM) reversibly increased the holding current, an effect mimicked by the prostanoid agonist iloprost (5–50 nM). The PGE 1 effect was totally blocked by the cAMP-antagonist Rp-cAMPS whereas H-89, an inhibitor of protein kinase A (PKA), failed to inhibit it, even when applied in the fairly high bath concentration of 30 μM. PGE 1 and iloprost also inhibited the T-type and hva Ca 2+ currents and this effect of PGE 1 was likewise not prevented by H-89. In some of the cells, the PGE 1 effect on holding current could be mimicked by 8-pCPT-2Me-cAMP (100–300 μM), a selective agonist of Epac (exchange protein activated by cAMP), but unlike the PGE 1 effect its action was not abolished by Rp-cAMPS. The effect of PGE 1 on the the holding current and on the T-type Ca 2+ current was diminished when EGTA in the pipette solution was replaced by BAPTA, suggesting that Ca 2+ ions are involved in the PGE 1 effect. It is concluded that the PGE 1 effect is mediated by cAMP and Ca 2+ ions but not by PKA or Epac.

Regulation by Prostaglandin E2 and Histamine of Angiogenesis in Inflammatory Granulation Tissue

AbstractFor Abstract see ChemInform Abstract in Full Text.

Regulation by prostaglandin E2 and histamine of angiogenesis in inflammatory granulation tissue.

In an air pouch-type carrageenin-induced inflammation model in rats, the selective cyclooxygenase (COX)-2 inhibitor NS-398 dose dependently inhibited the granulation tissue formation, angiogenesis and the level of vascular endothelial growth factor (VEGF) in the granulation tissue. In culture of the minced granulation tissue, PGE2 induced VEGF production in a concentration-dependent manner. Histamine also induced VEGF production in the granulation tissue in vitro. The H2 receptor antagonist cimetidine, the cAMP antagonist Rp-cAMP and the protein kinase A inhibitor H-89 suppressed the histamine-induced VEGF production in the granulation tissue. However, the H1 receptor antagonist pyrilamine maleate, the H3 receptor antagonist thioperamide, the protein kinase C inhibitors Ro 31-8425 and calphostin C or the tyrosine kinase inhibitor genistein showed no effect. Subcutaneous implantation of a cotton thread in the dorsum of histidine decarboxylase-deficient (HDC-/-) mice, but not in mast cell-deficient (WBB6F1-W/Wv) mice, induced less angiogenesis with lower levels of VEGF in the granulation tissue than in their corresponding wild-type (HDC+/+ and WBB6F1(-)+/+) mice. In HDC-/- mice, the topical injection of histamine or the H2 receptor agonist dimaprit rescued the defective angiogenesis and granulation tissue formation. In addition, cimetidine but not pyrilamine maleate and thioperamide inhibited the histamine-induced angiogenesis in the granulation tissue in HDC-/- mice. These findings suggest that PGE2 and histamine augment angiogenesis in the inflammatory granulation tissue by inducing VEGF production, and histamine induces VEGF production possibly through the H2 receptor--cAMP--protein kinase A pathway.

Roles of Prostaglandin E2 and Histamine in Angiogenesis in Inflammatory Granulation Tissue

In an air pouch-type carrageenin-induced inflammation model in rats, the selective cyclooxygenase (COX)-2 inhibitor NS-398 dose dependently inhibited the granulation tissue formation, angiogenesis and the level of vascular endothelial growth factor (VEGF) in the granulation tissue. In culture of the minced granulation tissue, PGE2 induced VEGF production in a concentration-dependent manner. Histamine also induced VEGF production in the granulation tissue in vitro. The H2 receptor antagonist cimetidine, the cAMP antagonist Rp-cAMP and the protein kinase A inhibitor H-89 suppressed the histamine-induced VEGF production in the granulation tissue. However, the H1 receptor antagonist pyrilamine maleate, the H3 receptor antagonist thioperamide, the protein kinase C inhibitors Ro31-8425 and calphostin C or the tyrosine kinase inhibitor genistein showed no effect. Subcutaneous implantation of a cotton thread in the dorsum of histidine decarboxylase-deficient (HDC-/-) mice, but not in mast cell-deficient (WBB6F1-W/Wv) mice, induced less angiogenesis with lower levels of VEGF in the granulation tissue than in their corresponding wild-type (HDC+/+ and WBB6F1-+/+) mice. In HDC-/- mice, the topical injection of histamine or the H2 receptor agonist dimaprit rescued the defective angiogenesis and granulation tissue formation. In addition, cimetidine but not pyrilamine maleate and thioperamide inhibited the histamineinduced angiogenesis in the granulation tissue in HDC-/- mice. These findings suggest that PGE2 and histamine play a significant roles in angiogenesis in the inflammatory granulation tissue via induction of VEGF production, and histamine augments VEGF production possibly through the H2 receptor-cAMP-protein kinase A pathway.

Pituitary Adenylate Cyclase-Activating Polypeptide Induces Astrocyte Differentiation of Precursor Cells from Developing Cerebral Cortex

Ciliary neurotrophic factor and bone morphogenetic proteins induce astrocytogenesis in the developing rat brain by stimulating STAT- and Smad-dependent signaling, respectively. We previously found that stimulation of the cAMP-dependent signaling pathway also triggers differentiation of cerebral cortical precursor cells into astrocytes, providing an additional mechanism to promote astrocyte differentiation. In this study, we show that pituitary adenylate cyclase-activating polypeptide (PACAP), but not the related vasoactive intestinal peptide, induces astrocyte differentiation of cortical precursor cells, even after a transient exposure. Cortical precursors were found to express predominantly the short isoform of the PACAP-specific PAC1 receptor, which couples to adenylate cyclase. Consistent with this notion, we determined that exposure of cortical precursors to PACAP resulted in a dose-dependent increase in cAMP production. Pretreatment of cells with the cAMP antagonist Rp-cAMPS prevented astrocyte differentiation. Thus, PACAP acts as an extracellular signal to trigger cortical precursor cell differentiation into astrocytes via stimulation of intracellular cAMP production.

Involvement of intracellular Ca2+ elevation but not cyclic AMP in PACAP-induced p38 MAP kinase activation in PC12 cells.

We have recently shown that in PC12 cells, pituitary adenylate cyclase-activating polypeptide (PACAP) and NGF synergistically stimulate PACAP mRNA expression primarily via a mechanism involving a p38 mitogen-activated protein kinase (MAPK)-dependent pathway. Here we have analyzed p38 MAPK activation by PACAP and the mechanism underlying this action of PACAP in PC12 cells. PACAP increased phosphorylation of p38 MAPK with a bell-shaped dose-response relationship and a maximal effect was obtained at 10(-8) M. PACAP (10(-8) M)-induced p38 MAPK phosphorylation was already evident at 2.5 min, maximal at 5 min, and rapidly declined thereafter. PACAP-induced p38 MAPK phosphorylation was potently inhibited by depletion of Ca(2+) stores with thapsigargin and partially inhibited by the phospholipase C inhibitor U-73122, L-type voltage-dependent calcium channel inhibitors nifedipine and nimodipine, and the Ca(2+) chelator EGTA, whereas the protein kinase C inhibitor calphostin C, the protein kinase A inhibitor H-89, the cAMP antagonist Rp-cAMP, and the nonselective cation channel blocker SKF96365 had no effect. These results indicate that PACAP activates p38 MAPK in PC12 cells through activation of a phospholipase C, mobilization of intracellular Ca(2+) stores, and Ca(2+) influx through voltage-dependent Ca(2+) channels, but not cyclic AMP-dependent mechanisms.

Parathyroid hormone activation of map kinase in rat duodenal cells is mediated by 3′,5′-cyclic AMP and Ca 2+

In a previous study, we demonstrated that parathyroid hormone (PTH) stimulates in rat duodenal cells (enterocytes) the phosphorylation and activity of extracellular signal-regulated mitogen-activated protein kinase (MAPK) isoforms ERK1 and ERK2. As PTH activates adenylyl cyclase (AC) and phospholipase C and increases intracellular Ca 2+ in these cells, in the present study we evaluated the involvement of cAMP, Ca 2+ and protein kinase C (PKC) on PTH-induced MAPK activation. We found that MAPK phosphorylation by the hormone did not depend on PKC activation. PTH response could, however, be mimicked by addition of forskolin (5–15 μM), an AC activator, or Sp-cAMP (50–100 μM), a cAMP agonist, and suppressed to a great extent by the AC inhibitor, compound Sq-22536 (0.2–0.4 mM) and the cAMP antagonist Rp-cAMP (0.2 mM). Removal of external Ca 2+ (EGTA 0.5 mM), chelation of intracellular Ca 2+ with BAPTA (5 μM), or blockade of L-type Ca 2+-channels with verapamil (10 μM) significantly decreased PTH-activation of MAPK. Furthermore, a similar degree of phosphorylation of MAPK was elicited by the Ca 2+ mobilizing agent thapsigargin, the Ca 2+ ionophore A23187, ionomycin and membrane depolarization with high K +. Inclusion of the calmodulin inhibitor fluphenazine (50 μM) did not prevent hormone effects on MAPK. Taken together, these results indicate that cAMP and Ca 2+ play a role upstream in the signaling mechanism leading to MAPK activation by PTH in rat enterocytes. As Ca 2+ and cAMP antagonists did not block totally PTH-induced MAPK phosphorylation, it is possible that linking of the hormone signal to the MAPK pathway may additionally involve Src, which has been previously shown to be rapidly activated by PTH. Of physiological significance, in agreement with the mitogenic role of the MAPK cascade, PTH increased enterocyte DNA synthesis, and this effect was blocked by the specific inhibitor of MAPK kinase (MEK) PD098059, indicating that hormone modulation of MAPK through these messenger systems stimulates duodenal cell proliferation.

Leptin Induces Mitochondrial Superoxide Production and Monocyte Chemoattractant Protein-1 Expression in Aortic Endothelial Cells by Increasing Fatty Acid Oxidation via Protein Kinase A

Leptin, a circulating hormone secreted mainly from adipose tissues, is involved in the control of body weight. The plasma concentrations are correlated with body mass index, and are reported to be high in patients with insulin resistance, which is one of the major risk factors for cardiovascular disease. However, the direct effect of leptin on vascular wall cells is not fully understood. In this study, we investigated the effects of leptin on reactive oxygen species (ROS) generation and expression of monocyte chemoattractant protein-1 (MCP-1) in bovine aortic endothelial cells (BAEC). We found that leptin increases ROS generation in BAEC in a dose-dependent manner and that its effects are additive with those of glucose. Rotenone, thenoyltrifluoroacetone (TTFA), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), uncoupling protein-1 (UCP1) HVJ-liposomes, or manganese superoxide dismutase (MnSOD) HVJ-liposomes completely prevented the effect of leptin, suggesting that ROS arise from mitochondrial electron transport. Leptin increased fatty acid oxidation by stimulating the activity of carnitine palmitoyltransferase-1 (CPT-1) and inhibiting that of acetyl-CoA carboxylase (ACC), pace-setting enzymes for fatty acid oxidation and synthesis, respectively. Leptin-induced ROS generation, CPT-1 activation, ACC inhibition, and MCP-1 overproduction were found to be completely prevented by either genistein, a tyrosine kinase inhibitor, H-89, a protein kinase A (PKA) inhibitor, or tetradecylglycidate, a CPT-1 inhibitor. Leptin activated PKA, and the effects of leptin were inhibited by the cAMP antagonist Rp-cAMPS. These results suggest that leptin induces ROS generation by increasing fatty acid oxidation via PKA activation, which may play an important role in the progression of atherosclerosis in insulin-resistant obese diabetic patients.

Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element.

Glucagon-like peptide 1 (GLP-1), a hormonal activator of adenyl cyclase, stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). Here we demonstrate that the signaling mechanism underlying stimulatory effects of GLP-1 on insulin gene transcription results from protein kinase A (PKA)-independent activation of the RIP1 CRE. Although GLP-1 stimulates cAMP production in rat INS-1 insulinoma cells, we find accompanying activation of a -410-bp RIP1 luciferase construct (-410RIP1-LUC) to exist independently of this second messenger. GLP-1 produced a dose-dependent stimulation of -410RIP1-LUC (EC50 0.43 nmol/l), an effect reproduced by the GLP-1 receptor agonist exendin-4 and abolished by the antagonist exendin(9-39). Activation of RIP1 by GLP-1 was not affected by cotransfection with dominant-negative Gs alpha, was not blocked by cAMP antagonist Rp-cAMPS, and was insensitive to PKA antagonist H-89. Truncation of -410RIP1-LUC to generate -307-, -206-, and -166-bp constructs revealed 2 segments of RIP1 targeted by GLP-1. The first segment, not regulated by forskolin, was located between -410 and -307 bp of the promoter. The second segment, regulated by both GLP-1 and forskolin, included the CRE and was located between -206 and -166 bp. Consistent with these observations, stimulatory effects of GLP-1 at RIP1 were reduced after introduction of delta-182 and delta-183/180 inactivating deletions at the CRE. The action of GLP-1 at -410RIP1-LUC was also reduced by cotransfection with A-CREB, a genetically engineered isoform of the CRE binding protein CREB, which dimerizes with and prevents binding of basic-region-leucine-zipper (bZIP) transcription factors to the CRE. In contrast, the action of GLP-1 at the CRE was not blocked by cotransfection with M1-CREB, an isoform that lacks a consensus serine residue serving as substrate for PKA-mediated phosphorylation. On the basis of these studies, it is proposed that PKA-independent stimulatory actions of GLP-1 at RIP1 are mediated by bZIP transcription factors related in structure but not identical to CREB.

Acute modulation of Ca 2+ influx on rat heart by 17β-estradiol

Estrogens initiate their action by binding to specific intracellular receptors and then acting on gene expression. In addition, there is growing evidence of a direct membrane effect via interaction with a cell surphase receptor. The aim of the present study was to investigate the acute effects of 17β-estradiol on Ca 2+ fluxes through second messenger pathways in rat cardiac muscle. Exposure of rat ventricle to low levels of 17β-estradiol (10 −12–10 −8 M) increased 45Ca 2+ influx within 1 min (+38%); the response was biphasic, peaking at 2 and 5 min (+60 and +55%, respectively). The effect of the hormone on rat heart seems to be specific since 17α-estradiol, dihydrotestosterone, and progesterone were devoid of activity. The effect of 17β-estradiol (5 min, 10 −10 M) was suppressed by nitrendipine (1 μM) and LaCl 3 (10 μM), involving the activation of voltage-dependent Ca 2+ channels in the acute increase of rat heart calcium influx by the hormone. 17β-estradiol rapidly increased cAMP content and PKA activity of rat cardiac muscle in parallel to the changes in Ca 2+ uptake. In addition the cAMP antagonist Rp-cAMPS suppressed 17β-estradiol-dependent Ca 2+ influx. Altogether, the data suggest the involvement of the cAMP/PKA messenger system in the nongenomic modulation of Ca 2+ influx in rat cardiac muscle by physiological levels of 17β-estradiol.

Acute increase, stimulated by prostaglandin E2, in glucose absorption via the sodium dependent glucose transporter-1 in rat intestine

BACKGROUND/AIMSAcute stimulation by cAMP of the sodium dependent glucose cotransporter SGLT1 has previously been shown. As prostaglandin E2(PGE2) increases intracellular cAMP concentrations via its receptor subtypes EP2R and EP4R, it...