Cyclase System Research Articles

Cardiotoxicity of β-mimetic catecholamines during ontogenetic development — possible risks of antenatal therapy

Catecholamines are involved in the regulation of a wide variety of vital functions. The β-adrenergic receptor (β-AR) - adenylyl cyclase system has been identified early in embryogenesis before the heart has received adrenergic innervation. The structure of β-receptors in the immature myocardium is similar to that in adults; there are, however, significant quantitative developmental changes in the inotropic and chronotropic responsiveness. Information on the toxic effect of the β-AR agonists in the immature heart is surprisingly scarce, even though these agents are used in clinical practice both during pregnancy and in early postnatal development. Large doses of β-AR agonists induce malformations of the cardiovascular system; the type of change depends upon the time at which the β-AR agonist was administered during embryogenesis. During postnatal ontogeny, the cardiotoxicity of β-AR agonists increased from birth to adulthood. It seems likely that despite interspecies differences, developmental changes in the cardiac sensitivity to β-AR agonists may exist in all mammals, depending on the degree of maturation of the system involved in β-adrenergic signaling. All the existing data draw attention to the possible harmful consequences of the clinical use of β-AR agonists during early phases of cardiac development. Late effects of the early disturbances of the cardiac muscle cannot be excluded.

Genetics of guanylyl cyclase pathways in the cochlea and their influence on hearing.

Although hearing loss is the most common sensory deficit in Western societies, there are no successful pharmacological treatments for this disorder. Recent experiments have demonstrated that manipulation of intracellular cyclic guanosine monophosphate (cGMP) concentrations can have both beneficial and harmful effects on hearing. In this review, we will examine the role of cGMP as a key second messenger involved in many aspects of cochlear function and discuss the known functions of downstream effectors of cGMP in sound processing. The nitric oxide-stimulated soluble guanylyl cyclase system (sGC) and the two natriuretic peptide-stimulated particulate GCs (pGCs) will be more extensively covered because they have been studied most thoroughly. The cochlear GC systems are attractive targets for medical interventions that improve hearing while simultaneously representing an under investigated source of sensorineural hearing loss.

Changes in Nociceptive Thresholds and Adenylyl Cyclase System Activity in Skeletal Muscles in Rats with Acute and Mild Type 1 Diabetes

Diabetic peripheral neuropathy (DPN) is one of the commonest complications of type 1 diabetes mellitus (DM1). The aims of the present work were to study the dynamics of the development of pain-type DPN and the functional status of the hormone-sensitive adenylyl cyclase signal system (ACSS) in the skeletal muscle of rats with models of acute and mild DM1 and to investigate the influences on these of insulin therapy using different routes of hormone administration – intranasal and peripheral. The nociceptive threshold in rats decreased in both models of DM1; the stimulatory effects of guanine nucleotides (GIDP) and adrenergic agonists (isoproterenol, BRL-37344) on adenylyl cyclase (AC) also decreased. The AC-stimulating effect of relaxin decreased in animals with acute DM1, while the change in mild DM1 was minor. Peripheral administration of insulin to rats with acute DM1 increased the nociceptive threshold and partially restored the AC effect of the β3 agonist BRL-37344. Intranasal administration of insulin to rats with mild DM1 also led to an increase in the nociceptive threshold and partially restored basal and BRL-37344-stimulated AC activity in the skeletal muscle of diabetic animals. Thus, skeletal muscle in rats with acute and mild DM1 showed impaired nociceptive sensitivity and ACSS function, which was partially restored by treatment of animals with insulin given peripherally (acute DM1) or intranasally (mild DM1).

Evaluation of Cytotoxicity and Hypoxic Effect of Nitroimidazole Embedded Nanoparticles.

Adenylate cyclase is a key intracellular enzyme involved in energy imbalance leading to tumor hypoxia and cytotoxicity. In this study, adenylate cyclase activities in isolated hepatocytes and Kupffer cells were compared in the presence of several metabolic stimulators. In cultured hepatocyte cells, adenylate cyclase was stimulated by guanylyl imidotriphosphate (GITP), guanosine triphosphate (GTP), progesterone and nitroimidazole embedded nanoparticle (NNP) effectors, while prostaglandin E2 and F2α were used as effectors in cultured Kupffer cells. The results showed that NNPs decreased adenylate cyclase specific activity in a dose-dependent manner after preincubation of hepatocytes with NNPs. The NNPs stimulated adenylate cyclase activities in hepatocytes were evaluated based on measurement of cyclic adenosine monophosphate (cAMP). The stimulatory effects of NNPs on adenylate cyclase were independent of the presence of GTP and may have been due to a direct effect on the catalytic subunit of adenylate cyclase. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the NNPs. In conclusion, NNPs exerted direct effects on the catalytic subunit of the adenylate cyclase system, and adenylate cyclase was hormone sensitive in liver cells.

Regulation of the Melanocortin-Sensitive Adenylate Cyclase System by N-Acylated Peptide 71-82 of Type 4 Melanocortin Receptor.

The peptides structurally corresponding in to cytoplasmic loops of G protein-coupled receptors (GPCR) are able to control functional activity of homologous receptors and the corresponding signaling pathways. Modification of these peptides with hydrophobic radicals enhances their biological activity due to penetration of lipophilic derivatives through the membrane and anchoring near their targets, GPCR. We synthesized an N-palmitoylated peptide Palm-Val-[Lys-Asn-Lys-Asn-Leu-His-Ser-Pro-(Nle)-Tyr-Phe-Phe71-82]-amide-Palm-Val-(71-82) structurally corresponding to cytoplasmic loop 1 of melanocortin 4 receptor (M4R). We found that in micromolar concentrations it very effectively suppresses stimulation of basal adenylate cyclase activity and basal level of GppNHp binding of heterotrimeric G proteins produced by THIQ and α-melanocyte stimulating hormone (α-MSH), agonists of M4R homologous to the peptide, in synaptosomal membranes of rat brain. The peptide Palm-Val-(71-82) also reduced, albeit to a significantly less extent, stimulation of adenylate cyclase and G-proteins by M3R agonist of γ-MSH, due to high homology of the peptide primary structure to M3R cytoplasmic loop 1. The synthesized peptide with activity of M4R/M3R antagonist can be used for the development of regulators of M4R and M3R and the corresponding biochemical and physiological processes.

The effect of prolonged metformin treatment on the activity of the adenylyl cyclase system and NO-synthase in the brain and myocardium of obese rats

The biguanide metformin, which is widely used for the treatment of type 2 diabetes, improves carbohydrate and lipid metabolism and has substantial cardioand neuroprotective effects. The positive effects of metformin on the activity of NO synthases catalyzing the synthesis of NO, a major vasodilator, and that of the hormone-sensitive adenylyl cyclase signaling system (ACSS) are believed to play an important role in the mediation of these effects. To prove this hypothesis, we initiated a study addressing the effect of prolonged metformin treatment of obese rats on metabolic parameters and the activity of ACSS and NO synthases in the myocardium and brain of the animals. Obesity was induced in Wistar rats by a high-fat diet, and the animals were subsequently treated with metformin for 2 months (the daily dose was 200 mg/kg). Metformin treatment induced a decrease in adipose tissue mass, total body mass, levels of insulin and glucose, and the insulin resistance index HOMA-IR, as well as improved glucose tolerance, and led to a decrease of the content of atherogenic forms of cholesterol. ACSS stimulation by agonists of β1/β2-adrenergic receptors (ARs) and enhancement of β3-AR signaling was detected in the myocardium of obese rats, while metformin treatment restored ACSS regulation by adrenergic agonists in the myocardium and normalized the balance between β-AR-signaling pathways. The stimulating effects of serotonin and agonists of type 4 melanocortin receptors on adenylyl cyclase, which are attenuated in obesity, were restored in the brain of rats treated with metformin. The treatment completely restored the total activity of NO synthases and the activity of endothelial NO synthase in the myocardium, which are reduced in obesity. Metformin treatment was also shown to induce hyperactivation of NO synthases in the brain and myocardium of healthy animals. It is proposed the effects of prolonged treatment of obese rats with metformin underlie the cardioand neuroprotective actions of this drug.

The functional activity of the adenylate cyclase system in the brains of rats with metabolic syndrome induced by immunization with peptide 11–25 of the type 4 melanocortin receptor

The melanocortin system of the brain, which includes melanocortin receptors of the fourth type (M4R), plays a key role in the regulation of energy homeostasis and controls functions of the nervous system. Inhibition of M4R results in obesity and the metabolic syndrome, which presumably occur due to changes in the neuromediator systems of the brain. To examine this hypothesis, we examined the effect of long-term immunization of rats with the BSA-conjugated K-[TSLHLWNRSSHGLHG11–25]-A peptide (K-[11–25]-A), which corresponds to the extracellular N-terminal domain of M4R, on the activity of the hormone-sensitive adenylate cyclase signaling system (ACSS) of the brain. In rats that were immunized numerous times with the BSA conjugate (the I group), we observed an increase in body weight, impaired glucose tolerance, insulin resistance, and dyslipidemia. At 13 months after the beginning of the experiment, we evaluated the ACSS activity in synaptosomal membranes from the brain. The basal activity of AC and its regulation by GppNHp and forskolin did not differ from the control. In the I group both the AC-stimulating effects of the α-melanocyte-stimulating hormone (α-MSH), M4R-agonist THIQ, dopamine, and pituitary AC-activating polypeptide and the AC-inhibiting effects of serotonin and 5-nonyloxytryptamine, an agonist of 5-hydroxytryptamine receptor (5-HTR) of the 1B/1D-subtype, decreased. The affinity of M4R to agonists did not change. The AC-stimulating effect of the M3R agonist γ-MSH was enhanced, which is a compensation for the weakening of M4R functions. The AC-stimulating effects of serotonin, EMD-386088, an agonist of 6 type 5-HTR relaxin, and noradrenaline, as well as the inhibitory effects of noradrenaline, the D2-agonist bromocriptine, and somatostatin in the I group did not change. Thus, inhibition of M4R as a result of immunization with the BSA conjugate of the K-[11–25]-A peptide alters the sensitivity of the ACSS of the rat brain to peptides of melanocortin family and other neurohormones. These alterations are characterized by hormonal and receptor specificity and may be one of the causes of insulin resistance, metabolic syndrome, and functional disturbances in the CNS and at the periphery under conditions of M4R deficit.

Changes in the nitric oxide-soluble guanylate cyclase system and natriuretic peptide receptor system in placentas of pregnant Dahl salt-sensitive rats.

Diminished vasodilator activity during pregnancy, which augments vascular responses to vasoconstrictors, is one reason for the onset of pre-eclampsia and superimposed pre-eclampsia. It is known that Dahl salt-sensitive (Dahl-S) rats develop salt-sensitive hypertension like African-Americans. The present study attempted to assess the changes and the interactions of the NOS-NO-sGC-cGMP and NP-NPR-cGMP systems in the hypertensive placenta using Dahl-S rats as an animal model of superimposed pre-eclampsia. Pregnant Dahl-S rats were fed a high-salt diet to induce the development of hypertension and fetal growth restriction. Using these rats, we investigated the regulation of these two vasodilatation systems, including the kinetics of cyclic guanosine monophosphate (cGMP), soluble guanylate cyclase (sGC), endothelial nitric oxide synthase (NOS), cytokine-inducible NOS, natriuretic peptides (NP) (atrial NP, brain NP and C-type NP), and NP receptors (NPR) (NPR-A, NPR-B, NPR-C). Dahl-S rats fed a high-salt diet exhibited hypertension, fetal growth restriction and thickening of the walls in decidual vessels. The placental cGMP level in the rats fed the high-salt diet was significantly decreased compared with that in controls. The expression levels of endothelial NOS and cytokine-inducible NOS mRNA increased significantly, while that of sGCα2-sunbnit declined significantly. Messenger RNA levels of NPR-C, a clearance-type receptor of NP, declined significantly, whereas those of NP and their functional receptors NPR-A and NPR-B were unchanged. As Dahl-S rats with excess salt-loading during pregnancy exhibited pathological changes similar to those observed in female humans with pre-eclampsia/superimposed pre-eclampsia, this rat could be useful as an animal model of superimposed pre-eclampsia. In the placentas of hypertensive Dahl-S rats, vasodilatation seemed to be disturbed by the deregulation of both the NO-sGC-cGMP and NP-NPR-cGMP systems.

The influence of bromocryptine treatment on activity of the adenylyl cyclase system in the brain of rats with type 2 diabetes mellitus induced by high-fat diet.

The influence of bromocryptine treatment on activity of the adenylyl cyclase system in the brain of rats with type 2 diabetes mellitus induced by high-fat diet.

Attenuation of inhibitory influence of hormones on adenylyl cyclase systems in the myocardium and brain of obese and type 2 diabetic rats as affected by the intranasal insulin treatment

The functional state of adenylyl cyclase signaling system (ACSS) and its regulation by the hormones and the inhibitor of adenylyl cyclase (AC), somatostatine (SST), in the brain and myocardium and by 5-nonyloxytryptamine (5-NOT) in the brain of rats of different ages (2- and 7-month-old) with experimental obesity and combination of obesity and type 2 diabetes mellitus (DM2), as well as the effect of the long-term treatment with intranasally administered insulin (II) on ACSS were studied. It was shown that the basal AC activity in obese and DM2 rats increases in the myocardium and, to a lesser extent, in the brain and decreases under the II treatment. The AC stimulating effect of forskolin decreases in the myocardium, but not in the brain of obese and DM2 rats. The II treatment recovers the AC stimulating effect of forskolin in 7-month-old animals, but has little effect in 5-month-old rats. In obesity as well as under the II treatment, the basal AC activity and its stimulation by forskolin change insignificantly. The AC inhibitory effects of 5-NOT and, particularly, SST are strongly attenuated in the investigated pathology, supposedly due to a reduction in the functional activity of Gi-proteins. The treatment of obesity and its combination with DM2 recovers, completely or partially, the AC inhibitory effects of hormones, most of all in the brain. Since the ACSS dysfunctions are causal to metabolic syndrome and DM2, their elimination by the II treatment promises an effective approach to combat these pathologies and their CNS and cardiovascular complications.

Aging of platelet nitric oxide signaling: pathogenesis, clinical implications, and therapeutics.

The nitric oxide (NO)/soluble guanylate cyclase (sGC) system is fundamental to endothelial control of vascular tone, but also plays a major role in the negative modulation of platelet aggregation. The phenomenon of platelet NO resistance, or decreased antiaggregatory response to NO, occurs increasingly with advanced age, as well as in the context of cardiovascular disease states such as heart failure, ischemic heart disease, and aortic valve disease. The central causes of NO resistance are "scavenging" of NO and dysfunction of sGC. In the current review, we discuss the roles of several modulators of NO synthesis and of the NO/sGC cascade on changes in platelet physiology with aging, together with potential therapeutic options to reduce associated thrombotic risk.

A Japanese family with nonautoimmune hyperthyroidism caused by a novel heterozygous thyrotropin receptor gene mutation

Hyperthyroidism caused by activating mutations of the thyrotropin receptor gene (TSHR) is rare in the pediatric population. We found a Japanese family with hyperthyroidism without autoantibody. DNA sequence analysis of TSHR was undertaken in this family. The functional consequences for the Gs-adenylyl cyclase and Gq/11-phospholipase C signaling pathways and cell surface expression of receptors were determined in vitro using transiently transfected human embryonic kidney 293 cells. We identified a heterozygous mutation (M453R) in exon 10 of TSHR. In this family, this mutation was found in all individuals who exhibited hyperthyroidism. The results showed that this mutation resulted in constitutive activation of the Gs-adenylyl cyclase system. However, this mutation also caused a reduction in the activation capacity of the Gq/11-phospholipase C pathway, compared with the wild type. We demonstrate that the M453R mutation is the cause of nonautoimmune hyperthyroidism.

Regulation of adenylyl cyclase activity in rat testes by acylated derivatives of peptide 562–572 of a luteinizing hormone receptor

One of directions of the search of hormonal signaling systems regulators is the development of peptides that correspond to the cytoplasmic regions of G-protein-coupled receptors (GPCR). Modification of the pepti- des with hydrophobic radicals increases their efficiency and selectivity. But currently is not studied as the acti- vity of the peptide depends on the localization of the hydrophobic radicals, their number and the chemical natu- re. The aim of this work was the synthesis of modified by fatty acid radicals derivatives of peptide 562-572 corresponding to the C-terminal region of luteinizing hormone receptor (LHR), and the study of regulatory ef- fects of the acylated LHR-peptides on the basal and hormone-stimulated activity of adenylyl cyclase (AC) in the rat tissues. To elucidate the effect of localization of hydrophobic radicals and their number the modification of peptide 562-572 only at the N- or C-terminus or at both ends was carried out. To study the effect of hydrop- hobicity the residues ofpalmitic (Pal) and decanoic (Dec) acids were selected. Using a solid phase strategy we have synthesized unmodified peptide NDTKIAKK-Nle-A562-572-KA (1) and five of its acylated analogues, such as N[K(Dec)]DTKIAKK-Nle-A562-572-KA (2), NKDTKIAKK-Nle-A562-572-[K(Dec)]A (3), N[K(Dec)] DTKIAKK-Nle-A562-572-[K(Dec)]A (4), N[K(Pal)]DTKIAKK-Nle-A562-572-KA (5), and NKDTKIAKK-Nle- A562--572-[K(Pal)]A (6). Peptide 6 modified with palmitate at the C-terminus to a large extent increased the ba- sal AC activity and reduced AC stimulating effect of human chorionic gonadotropin (hCG) in the testes of rats, peptides 3 and 4 modified with decanoate at the C-terminus were less effective, but exceeded in activity the un- modified peptide 1, while peptides 2 and 5 acylated at the N-terminus were little active. The action of peptides was characterized by the tissue and the receptor specificity. Thus, the modification of LHR-peptide 562-572 with fatty acid radicals at the C-terminus increases its regulatory effect on the functional activity of the adenyla- te cyclase system in the rat testes, indicating promising the modification of GPCR-peptides with hydrophobic radicals. These data support the hypothesis that the hydrophobic radical to be localized in the locus of GPCR-peptide, where a transmembrane domain is located in the receptor.

β-adrenergic receptor responsiveness in aging heart and clinical implications.

Elderly healthy individuals have a reduced exercise tolerance and a decreased left ventricle inotropic reserve related to increased vascular afterload, arterial-ventricular load mismatching, physical deconditioning and impaired autonomic regulation (the so called “β-adrenergic desensitization”). Adrenergic responsiveness is altered with aging and the age-related changes are limited to the β-adrenergic receptor density reduction and to the β-adrenoceptor-G-protein(s)-adenylyl cyclase system abnormalities, while the type and level of abnormalities change with species and tissues. Epidemiological studies have shown an high incidence and prevalence of heart failure in the elderly and a great body of evidence correlate the changes of β-adrenergic system with heart failure pathogenesis. In particular it is well known that: (a) levels of cathecolamines are directly correlated with mortality and functional status in heart failure, (b) β1-adrenergic receptor subtype is down-regulated in heart failure, (c) heart failure-dependent cardiac adrenergic responsiveness reduction is related to changes in G proteins activity. In this review we focus on the cardiovascular β-adrenergic changes involvement in the aging process and on similarities and differences between aging heart and heart failure.

Low and High Temperatures Enhance Guanylyl Cyclase Activity in Arabidopsis Seedlings

Low and High Temperatures Enhance Guanylyl Cyclase Activity in Arabidopsis Seedlings Guanylyl cyclase system plays a crucial role in intracellular signaling in animals and plants involving cyclic guanosine 3’,5’-monophosphate (cGMP) as a signaling intermediate. cGMP is a well-established intracellular molecule that takes part in diverse physiological processes, biotic and abiotic stress responses in higher plants. We have focused here on participation of cGMP as a secondary messenger in temperature stress-induced pathways and the mechanism of cGMP-mediated signal transduction in Arabidopsis seedlings.

Relationship between the changes in peripheral thresholds of nociception and activity of the adenylyl cyclase system in the skeletal muscles of rats with streptozotocin-induced diabetes

Relationship between the changes in peripheral thresholds of nociception and activity of the adenylyl cyclase system in the skeletal muscles of rats with streptozotocin-induced diabetes

The effect of long-term diabetes mellitus induced by treatment with streptozotocin in 6-week-old rats on functional activity of the adenylyl cyclase system

In type 1 diabetes mellitus (DM), changes occurring in the adenylyl cyclase signaling system (ACSS) are one of the key causes of complications of the disease. Since type 1 DM has been most often diagnosed in childhood and adolescence, the study of changes in ACSS in the early development of the disease is a genuine problem. For this, we developed a prolonged model of type 1 DM, which was induced by treatment of 6-week-old rats with moderate doses of streptozotocin (1.5M-DM), and studied the functional state of ACSS in the brain, myocardium, and testes of rats with this model of the disease 7 months after its start. The 1.5M-DM model was compared with the model that was induced by streptozotocin treatment of adult, 5-month-old animals (5M-DM). It was shown that, in 1.5M-DM, in the tissues of diabetic rats, the functional activity of ACSS sensitive to biogenic amines and polypeptide hormones was significantly changed. In rats with 1.5M-DM, the adenylate cyclase (AC) inhibitory effects of somatostatin (in all studied tissues), noradrenaline (in the myocardium and the brain), and agonists of type 1 serotonin receptor (in the brain) were weakened to the greatest degree. In the brain, the AC-stimulating effects of relaxin, isoproterenol, and agonists of Gs-protein-coupled serotonin receptors also decreased; in the myocardium, the corresponding effects of GppNHp, relaxin, and β-adrenergic agonists declined; and, in the testes, the AC effects of GppNHp and chorionic gonadotropin declined. When comparing the 1.5M-DM and 5M-DM models, the most pronounced differences between them were found in the effect of DM on hormonal regulation of ACSS in the brain, this being true both for AC-stimulating effects of dopamine and PACAP-38 and for AC-inhibiting effects of bromocriptine and somatostatin. These results indicate significant changes in hormonal regulation of the nervous, cardiovascular, and reproductive systems in rats with early induction of type 1 DM, in some cases more severe changes as compared with late model of 5M-DM. These changes may be the basis for development of diabetic cardiomyopathy, cognitive deficiency, and hypogonadotropic states, which are often detected in children and adolescents with type 1 DM.

Interactions Affected by Arginine Methylation in the Yeast Protein–Protein Interaction Network

Protein-protein interactions can be modulated by the methylation of arginine residues. As a means of testing this, we recently described a conditional two-hybrid system, based on the bacterial adenylate cyclase (BACTH) system. Here, we have used this conditional two-hybrid system to explore the effect of arginine methylation in modulating protein-protein interactions in a subset of the Saccharomyces cerevisiae arginine methylproteome network. Interactions between the yeast hub protein Npl3 and yeast proteins Air2, Ded1, Gbp2, Snp1, and Yra1 were first validated in the absence of methylation. The major yeast arginine methyltransferase Hmt1 was subsequently included in the conditional two-hybrid assay, initially to determine the degree of methylation that occurs. Proteins Snp1 and Yra1 were confirmed as Hmt1 substrates, with five and two novel arginine methylation sites mapped by ETD LC-MS/MS on these proteins, respectively. Proteins Ded1 and Gbp2, previously predicted but not confirmed as substrates of Hmt1, were also found to be methylated with five and seven sites mapped respectively. Air2 was found to be a novel substrate of Hmt1 with two sites mapped. Finally, we investigated the interactions of Npl3 with the five interaction partners in the presence of active Hmt1 and in the presence of Hmt1 with a G68R inactivation mutation. We found that the interaction between Npl3 and Air2, and Npl3 and Ded1, were significantly increased in the presence of active Hmt1; the interaction of Npl3 and Snp1 showed a similar degree of increase in interaction but this was not statistically significant. The interactions of Npl3 and Gbp2, along with Npl3 and Yra1, were not significantly increased or decreased by methylation. We conclude that methylarginine may be a widespread means by which the interactions of proteins are modulated.

Enterotoxigenic Escherichia coli infection and intestinal thiamin uptake: studies with intestinal epithelial Caco-2 monolayers

Infections with enteric pathogens like enterotoxigenic Escherichia coli (ETEC) is a major health issue worldwide and while diarrhea is the major problem, prolonged, severe, and dual infections with multiple pathogens may also compromise the nutritional status of the infected individuals. There is almost nothing currently known about the effect of ETEC infection on intestinal absorptions of water-soluble vitamins including thiamin. We examined the effect of ETEC infection on intestinal uptake of the thiamin using as a model the human-derived intestinal epithelial Caco-2 cells. The results showed that infecting confluent Caco-2 monolayers with live ETEC (but not with boiled/killed ETEC or nonpathogenic E. coli) or treatment with bacterial culture supernatant led to a significant inhibition in thiamin uptake. This inhibition appears to be caused by a heat-labile and -secreted ETEC component and is mediated via activation of the epithelial adenylate cyclase system. The inhibition in thiamin uptake by ETEC was associated with a significant reduction in expression of human thiamin transporter-1 and -2 (hTHTR1 and hTHTR2) at the protein and mRNA levels as well as in the activity of the SLC19A2 and SLC19A3 promoters. Dual infection of Caco-2 cells with ETEC and EPEC (enteropathogenic E. coli) led to compounded inhibition in intestinal thiamin uptake. These results show for the first time that infection of human intestinal epithelial cells with ETEC causes a significant inhibition in intestinal thiamin uptake. This inhibition is mediated by a secreted heat-labile toxin and is associated with a decrease in the expression of intestinal thiamin transporters.

The roles of cyclic AMP–ERK–Bad signaling pathways on 6-hydroxydopamine-induced cell survival and death in PC12 cells

The roles of cyclic AMP (cAMP)–ERK1/2–Bad signaling pathways in 6-hydroxydopamine (6-OHDA)-induced cell survival and death were investigated. In PC12 cells, 6-OHDA (10–100μM) concentration-dependently increased the intracellular levels of cAMP mediated by the Ca2+-CaMKII-adenylyl cyclase system. 6-OHDA at the non-toxic level (10μM) induced transient ERK1/2 phosphorylation and BadSer112 phosphorylation, which maintained cell survival. In contrast, the high levels of cAMP induced by toxic levels (50 and 100μM) of 6-OHDA induced sustained ERK1/2 phosphorylaton and BadSer155 phosphorylation. The cells then moved to cell death process through Bcl2 phosphorylation and caspase-3 activation. BadSer155 phosphorylation by 6-OHDA was inhibited by PKA (H89) and MEK (U0126) inhibitors, indicating that it was mediated via the cAMP–PKA-sustained ERK1/2 system. In SK-N-BE(2)C cells, the non-toxic level of 6-OHDA also showed transient ERK1/2 phosphorylation and BadSer112 phosphorylation, and toxic levels of 6-OHDA exhibited sustained ERK1/2 phosphorylation and BadSer155 phosphorylation. These results suggest that ERK1/2 phosphorylation by 6-OHDA shows biphasic functions on cell survival and death in PC12 cells. It is, therefore, proposed that the cAMP–ERK1/2–Bad signaling pathways incurred by toxic levels of 6-OHDA play a role in dopamine neuron death of animal models of Parkinson’s disease.