Adenylyl Cyclase System Research Articles

The Brain Adenylyl Cyclase Signaling System and Cognitive Functions in Rats with Neonatal Diabetes under the Influence of Intranasal Serotonin

Type 2 diabetes mellitus is often associated with the neurodegenerative changes, and this is regarded as being the cause of cognitive deficit and other brain dysfunctions. The data obtained by us and the other authors showed that alterations in the brain signaling systems regulated by different hormones and neurotransmitters contribute to triggering and development of neurodegenerative processes in diabetes. However, the activity of brain adenylyl cyclase system and the cognitive functions, their interrelation, and the influence of intranasal serotonin on them in type 2 diabetes are poorly understood yet. We investigated the hormonal sensitivity of adenylyl cyclase system in the brain of female rats with the neonatal model of type 2 diabetes and the influence of 8-weeks treatment with intranasal serotonin (20 μ g/rat daily) on the brain adenylyl cyclase system and cognition in diabetes. It was shown that in the diabetic brain the regulatory effects of hormones (relaxin, adrenergic agonists, dopamine, serotonin) activating adenylyl cyclase via Gs proteins changed in a receptor-specific manner and were restored by intranasal serotonin. The effects of hormones inhibiting adenylyl cyclase via Gi proteins were significantly decreased, especially in the case of agonists of type 1 serotonin receptors. The intranasal serotonin treatment led to their partial or complete restoration. Using Morris water maze test we showed that intranasal serotonin improves diabetes- associated impaired learning and spatial memory. Summing up, in the brain of diabetic rats the functional activity of hormone-sensitive adenylyl cyclase signaling system was altered, most dramatically in the G i -coupled cascades. The intranasal serotonin treatment improved both the signal transduction via the brain adenylyl cyclase system and cognitive functions in type 2 diabetes.

Development of non-hormonal regulators of adenylyl cyclase signaling system on the basis of peptides, derivatives of the third intracellular loop of somatostatin receptors

In the majority of the serpentine type receptors the third intracellular loop (ICL-3) is responsible for interaction with heterotrimeric G-proteins and for transduction of hormonal signal to the enzymes, generators of the second messengers. It was found that the peptides corresponding to ICL-3 influence functional activity of hormonal signaling systems in the absence of the hormone and, in consequence, can be considered as prototypes for the development of selective regulators of these systems. We have originally synthesized peptides corresponding to C-terminal regions 255-269 and 240 254 of ICL-3 of type 1 and 2 rat somatostatin receptors (Som1R and Som2R). Micromolar concentrations of these peptides activated Gi-proteins and inhibited forskolin-stimulated activity of adenylyl cyclase (AC) in rat brain tissues. The peptide 255-269 of Som1R is a selective antagonist of Som1R, and the peptide 240-254 of Som2R is an agonist of Som1R. So, the peptide 255-269 of Som1R decreased the regulatory effects of somatostatin and selective Som1R-agonist CH-275 realized via the receptor homologous to them, while the peptide 240-254 of Som2R, on the contrary, increased AC inhibitory action of CH-275. Both peptides insignificantly influenced regulatory effects of the Som2R-agonist octreotide. Summing up, the peptides studied by us are selective regulators of somatostatin-sensitive AC system. Using the peptides it was shown that ICL-3 of Som1R and Som2R includes the main molecular determinants that are responsible for activation of Gi-proteins and regulation of AC system by somatostatin and its analogues.

Intranasal insulin affects adenyl cyclase system in rat tissues in neonatal diabetes

Abstract The changes in hormone-regulated adenylyl cyclase (AC) signaling system implicated in control of the nervous, cardiovascular and reproductive systems may contribute to complications of diabetes mellitus (DM). We investigated the functional state of AC system in the brain, myocardium, ovary and uterus of rats with neonatal DM and examined the influence of intranasally administered insulin on the sensitivity of this system to biogenic amines and polypeptide hormones. The regulatory effects of somatostatin and 5-HT1BR-agonist 5-nonyloxytryptamine acting via Gi protein-coupled receptors were significantly decreased in DM and partially restored in insulin-treated rats. The effects of hormones, activators of AC, are changed in tissue- and receptorspecific manner, and intranasal insulin restored the effects rather close to the level in control. In insulin-treated non-diabetic rats, AC stimulating effects of isoproterenol and relaxin in the myocardium and of human chorionic gonadotropin in the ovaries were decreased, while the effects of hormones, inhibitors of AC, were increased. These data indicate that with intranasal insulin, Gi protein-mediated signaling pathways continue to gain strength. The obtained data on the influence of hormones on AC system in the brain, myocardium, ovary and uterus allow looking anew into the mechanisms of therapeutic effects of intranasal insulin.

Gene expression profiling of pituitary melanotrope cells during their physiological activation

The pituitary melanotrope cells of the amphibian Xenopus laevis are responsible for the production of the pigment-dispersing peptide α-melanophore-stimulating hormone, which allows the animal to adapt its skin color to its environment. During adaptation to a dark background the melanotrope cells undergo remarkable changes characterized by dramatic increases in cell size and secretory activity. In this study we performed microarray mRNA expression profiling to identify genes important to melanotrope activation and growth. We show a strong increase in the expression of the immediate early gene (IEG) c-Fos and of the brain-derived neurotrophic factor gene (BDNF). Furthermore, we demonstrate the involvement of another IEG in the adaptation process, Nur77, and conclude from in vitro experiments that the expression of both c-Fos and Nur77 are partially regulated by the adenylyl cyclase system and calcium ions. In addition, we found a steady up-regulation of Ras-like product during the adaptation process, possibly evoked by BDNF/TrkB signaling. Finally, the gene encoding the 105-kDa heat shock protein HSPh1 was transiently up-regulated in the course of black-background adaptation and a gene product homologous to ferritin (ferritin-like product) was >100-fold up-regulated in fully black-adapted animals. We suggest that these latter two genes are induced in response to cellular stress and that they may be involved in changing the mode of mRNA translation required to meet the increased demand for de novo protein synthesis. Together, our results show that microarray analysis is a valuable approach to identify the genes responsible for generating coordinated responses in physiologically activated cells.

Development of non-hormonal regulators of the adenylyl cyclase signaling system based on the peptides, derivatives of the third intracellular loop of somatostatin receptors

In most serpentine type receptors the third intracellular loop (ICL-3) is responsible for the interaction with heterotrimeric G proteins and for transduction of a hormonal signal to the enzymes, generators of the second messengers. It was found that the peptides corresponding to ICL-3 influence functional activity of hormonal signaling systems in the absence of the hormone and, consequently, may be considered as prototypes for the development of selective regulators of these systems. We have originally synthesized peptides corresponding to the C-terminal regions 255–269 and 240–254 of ICL-3 of type 1 and 2 rat somatostatin receptors (Som1R and Som2R). Micromolar concentrations of these peptides activated Gi proteins and inhibited forskolin-stimulated activity of adenylyl cyclase (AC) in rat brain tissues. The peptide 255–269 of Som1R is a selective antagonist of Som1R, and the peptide 240–254 of Som2R is an agonist of Som1R. The peptide 255–269 of Som1R decreased the regulatory effects of somatostatin and the selective Som1R agonist, CH-275, realized via the homologous receptor, while the peptide 240–254 of Som2R, on the contrary, increased the AC inhibitory effect of CH-275. Both peptides insignificantly influenced regulatory effects of the Som2R agonist octreotide. Thus, the peptides studied by us are selective regulators of the somatostatin-sensitive AC system. Using the peptides we have demonstrated that ICL-3 of both Som1R and Som2R includes the main molecular determinants that are responsible for activation of Gi proteins and regulation of the AC system by somatostatin and its analogues.

A decrease in the sensitivity of adenylyl cyclase and heterotrimeric g proteins to chorionic gonadotrophin and peptide hormones action in the tissues of reproductive system of rats with experimental type 2 diabetes

Patients with different forms of the diabetes, particularly with insulin-independent type 2 diabetes, have a wide spectrum of the disturbances of the functions of reproductive system. It is suggested that the main reason of these disturbances is altered sensitivity of reproductive system tissues to the regulatory action of hormones. The aim of this study was the identification of the changes in functioning of adenylyl cyclase system (ACS) sensitive to human chorionic gonadotrophin (hCG) and the peptide hormones in the ovary, testes and uterus of rats with neonatal streptozotocin (STZ) diabetes that is similar to the type 2 diabetes in humans. The effects of hCG, PACAP-38 and relaxin, realizing their effects via stimulatory G proteins (Gs), and somatostatin, acting via the inhibitory G protein (Gi), on adenylyl cyclase (AC) activity and GTP binding to the G proteins were studied. In rats with STZ type 2 diabetes the regulatory effects of hCG and PACAP-38 decreased in the ovary and testes, while the effects of somatostatin decreased in all investigated tissues (especially in the uterus). This caused attenuation of the hormonal effects, stimulating (hCG and PACAP-38) or inhibiting (somatostatin) AC activity, and in the decrease of their stimulatory effect on the GTP binding. At the same time a significant decrease of ACS sensitivity to relaxin in the tissues of diabetic rats was not found. Data obtained suggest that one of the key reasons for impairments of reproductive functions in experimental type 2 diabetes is the decrease of ACS sensitivity to the hormones, hCG, PACAP-38 and somatostatin, which play an important role in the reproductive system functioning.

Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system in the infusorian Dileptus anser

In some unicellular eukaryotes, cAMP performs functions not only of the second messenger, but also of hormone, the primary messenger. We have found that cAMP binds to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and enzyme adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K(D), 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N6,O2-dibutyryl-cAMP, a non-hydrolyzed cAMP analog, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated with the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.

The Peptides Mimicking the Third Intracellular Loop of 5-Hydroxytryptamine Receptors of the Types 1B and 6 Selectively Activate G Proteins and Receptor-Specifically Inhibit Serotonin Signaling via the Adenylyl Cyclase System

The third intracellular loop (ICL3) of G protein-coupled receptors has, as a rule, a key role in their interaction with heterotrimeric G proteins. We synthesized peptides corresponding to the C-terminal region of the ICL3 (C-ICL3) of 5-hydroxytryptamine receptors of the type 1B (5-HT1BR) and 6 (5-HT6R) and studied their influence on the functional activity of adenylyl cyclase signaling system (ACSS) in synaptosomal membranes isolated from the rat brain. The 5-HT1BR-peptide ARERKATKTL307–316K-amide mimicking agonist-activated 5-HT1BR reduced forskolin-stimulated adenylyl cyclase (AC) activity and activated pertussis toxin-sensitive G proteins. It lowered inhibitory effects of serotonin and 5-HT1BR-agonists on forskolin-stimulated AC activity and their stimulating effects on GTP binding. This was not the case in the presence of 5-HT1BR-antagonists. The 5-HT6R-peptides mimicking 5-HT6R activated both the basal AC activity and GTP binding of cholera toxin-sensitive G proteins. They lowered the stimulating effect of serotonin and 5-HT6R-agonists on AC and Gs proteins, but in the presence of 5-HT6R-antagonists their action was blocked. Of all the 5-HT6R-peptides with linear and dimeric structure we studied the palmitoylated peptide KHSRKALKASL258–268K(Pal)A-amide had a most pronounced effect both on the basal and 5-HT6R-agonist-stimulated ACSS. The data was obtained indicating that the peptides corresponding to C-ICL3 of 5-HT1BR and 5-HT6R selectively activate Gi and Gs proteins, respectively, and in a receptor-specific manner reduce signal transduction via serotonin-sensitive ACSS in the rat brain. The results of the study give strong evidence in favor of active participation of C-ICL3 of these 5-HTRs in their coupling with the G proteins.

Inhibition of functional activity of the adenylyl cyclase signaling system of the ciliate Dileptus anser by colchicine and vinblastine

Cytoskeleton plays a key role in the functioning of hormonal signaling systems in vertebrate animals. However, data on the effect of cytoskeletal components, in particular microtubules, on the functional activity of chemosignaling systems of unicellular organisms are currently lacking. The goal of this work consisted of studying the effects of microtubule-disrupting agents, colchicine and vinblastine, on the adenylyl cyclase system of free living infusoria Dileptus anser. The incubation of D. anser with colchicine and vinblastine (10−5–10−6 M) weakly affected the basal activity of adenylyl cyclase (AC), but led to a significant decrease in or complete block of AC stimulation with nonhormonal (GppNHp, sodium fluoride) and hormonal agents (adrenaline, serotonin, glucagon). The basal level of GTP binding in heterotrimeric G proteins decreased and there was observed inhibition of stimulation of G proteins by hormones. Colchicine and vinblastine have been shown to interrupt adrenalin-produced AC stimulation achieved through Gs-protein, but weakly affect its inhibiting AC effect caused by the Gi-protein. Thus, it has been established for the first time that, in unicellular organisms, i.e., infusoria D. anser, microtubules are involved in the regulation of the functional activity of the AC system and their action is realized at the level of G proteins, which is similar to Gs-proteins in vertebrate animals.

A decrease of sensitivity of adenylyl cyclase and heterotrimeric g-proteins to chorionic gonadotropin and peptide hormones action in the tissues of reproductive system of the rats in the condition of experimental type 2 diabetes

Patients with different forms of the diabetes, particularly with insulin-independent type 2 diabetes have a wide spectrum of the disturbances of the functions of reproductive system. It is supposed that the main reason of these disturbances is altered sensitivity of reproductive system tissues to regulatory action of hormones. The aim of the work was the identification of the changes in functioning of human chorionic gonadotropin (hCG)--and peptide hormones-sensitive adenylyl cyclase system (ACS) in the ovary, testes and uterus of rats with neonatal streptozotocin (STZ) diabetes that is similar to the type 2 diabetes in humans. The effects of hCG, PACAP-38 and relaxin, realizing their effects via G-protein of the stimulatory type (Gs), and somatostatin, acting via G-protein of the inhibitory type (Gi), on adenylyl cyclase (AC) activity and the GTP binding of the G-proteins were studied. Regulatory effects of hCG and PACAP-38 decreased in the ovary and testes of rats with STZ type 2 diabetes, while the effects of somatostatin decreased in all investigated tissues (in a considerable extent in the uterus). This expressed in the weakening of hormonal effects on AC activity, stimulating in the case of hCG and PACAP-38 and inhibiting in the case of somatostatin, and in the decrease of stimulation of the GTP binding by the hormones. At the same time a significant decrease of ACS sensitivity to relaxin in the tissues of diabetic rats was not found. Data obtained suggest that the key reason of the disturbances of reproductive functions in experimental type 2 diabetes is the decrease of ACS sensitivity to the hormones, such as hCG, PACAP-38 and somatostatin, that play a important role in functioning of reproductive system.

CAMP-synthesis in a medullary thyroid carcinoma cell line: response to adrenergic agents and prostaglandines.

Calcitonin secretion by C-cells is mediated through intracellular 3'5'-cyclic adenosine monophosphate (cAMP) and calcium signaling. Calcitonin release stimulation tests may take advantage of both signaling cascades in screening for medullary thyroid carcinomas (MTC). To elucidate the regulation of the adenylyl cyclase system we have determined cAMP levels of a calcitonin-expressing MTC cell line (RG) after exposure to adrenergic agents and prostaglandines. In early passages (20-30) cAMP concentrations were significantly elevated in RG cells after exposure to beta-adrenergic agents and prostaglandines E1 and E2. In advanced passages (60-80) the beta-adrenergic response was no longer detectable and adrenergic receptors were uncoupled from the adenylyl cyclase complex; while the effect of prostaglandines E1 and E2 remained unaffected. Preincubation with dexamethasone, in a process requiring protein new synthesis, re-established the adrenergic response in later passages, indicating that RG cells dedifferentiated in culture over time. Our in vitro findings suggest that MTC cell dedifferentiation may be accompanied by adrenergic receptor-uncoupling from the adenylate cyclase system and that this process may be reversed by dexamethasone incubation.

Receptor-independent sensitization of the adenylyl cylase after chronic treatment with cyclosporine A

Chronic treatment with cyclosporine A (CyA) is often complicated by severe hypertension. If activation of the beta-adrenergic-receptor-linked adenylyl cyclase (AC) system contributes to hypertension is unresolved. Rats were treated with CyA (20 mg kg(-1) day(-1)) for 7 days. beta-adrenergic, muscarinic, and alpha-adrenergic receptors, G-proteins, and the activity of AC were determined in cardiac and pulmonary plasma membranes. The density of cardiac beta-adrenergic receptors, muscarinic receptors, alpha-adrenergic receptors, G(alphas) and, G(alphai) remained unchanged after treatment with CyA. However, CyA increased the responsiveness of AC to different stimulators. The responsiveness of AC was even more pronounced after solubilization and partial purification, suggesting a direct modulation of the enzyme. These data suggest that CyA modulates the activity of the sympathoadrenergic system by a direct, receptor-independent sensitization of AC, suggesting that this pathway contributes to hypertension in patients treated with CyA.

Vitamin B12deficiency results in the abnormal regulation of serine dehydratase and tyrosine aminotransferase activities correlated with impairment of the adenylyl cyclase system in rat liver

The aim of the present study was to elucidate the mechanism of the vitamin B(12) deficiency-induced changes of the serine dehydratase (SDH) and tyrosine aminotransferase (TAT) activities in the rat liver. When rats were maintained on a vitamin B(12)-deficient diet, the activities of these two enzymes in the liver were significantly reduced compared with those in the B12-sufficient control rats (SDH 2.8 (sd 0.56) v. 17.5 (sd 6.22) nmol/mg protein per min (n 5); P < 0.05) (TAT 25.2 (sd 5.22) v. 41.3 (sd 8.11) nmol/mg protein per min (n 5); P < 0.05). In the B(12)-deficient rats, the level of SDH induction in response to the administration of glucagon and dexamethasone was significantly lower than in the B(12)-sufficient controls. Dexamethasone induced a significant increase in TAT activity in the primary culture of the hepatocytes prepared from the deficient rats, as well as in the cells from the control rats. However, a further increase in TAT activity was not observed in the hepatocytes from the deficient rats, in contrast to the cells from the controls, when glucagon was added simultaneously with dexamethasone. The glucagon-stimulated production of cAMP was significantly reduced in the hepatocytes from the deficient rats relative to the cells from the control rats. Furthermore, the glucagon-stimulated adenylyl cyclase activity in the liver was significantly lower in the deficient rats than in the controls. These results suggest that vitamin B(12) deficiency results in decreases in SDH and TAT activities correlated with the impairment of the glucagon signal transduction through the activation of the adenylyl cyclase system in the liver.

Disturbance of transduction of adenylyl cyclase-inhibiting hormonal signaling in the myocardium and brain of rats with experimental type 2 diabetes

Presently, our work, as well as that of other authors, has produced convincing evidence in favor of the idea that disturbances in hormonal signaling systems are one of the main causes of the development of pathological alterations and complications in diabetes. However, the molecular mechanisms underlying these disturbances remain practically unstudied, particularly in insulin-independent type 2 diabetes. Using a neonatal streptozotocin model of type 2 diabetes, whose duration was either 80 or 180 days, we studied changes in the functional activity of components of the hormone-regulated adenylyl cyclase (AC) signaling system in the myocardium and brain striatum of diabetic rats as compared with control animals. In diabetes, the Gi-realized process of transduction of the hormonal signal inhibiting AC activity has been shown to be markedly impaired. This is manifested as a decrease of the inhibitory effect of hormones on AC activity and an attenuation of their stimulation of the G-protein’s GTP-binding activity. In the case of noradrenaline (myocardium), the inhibitory pathway of the AC system regulation is completely suppressed, while the stimulatory pathway is preserved. An increase in the duration of diabetes development from 80 to 180 days leads to some decrease in the transduction of hormonal signals realized via Gi-proteins. The stimulatory effects of biogenic amines and relaxin on AC activity and GTP binding in the myocardium and brain of diabetic rats change relatively little, both in the 80-and in the 180-day diabetes. Thus, in the experimental type 2 diabetes, disturbances in Gi-protein coupled signal cascades are primarily observed, through which hormones realize their inhibition of AC activity.

Perilipin regulates the thermogenic actions of norepinephrine in brown adipose tissue

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.

Regulatory calcium effect on adenylyl cyclase functional activity in the infusorian Dileptis anser

Earlier we have shown that some non-hormonal activators of adenylyl cyclase (AC) and hormones of higher vertebrate animals are able to affect functional activity of the AC system in the infusorian Dileptus anser. In the present work, sensitivity of this infusorian AC to Ca2+ was studied and it was found that calcium cations at concentrations of 0.5-10 microM stimulated significantly the enzyme activity in D. anser partially purified membranes. An increase of Ca2+ concentrations to 100 microM and higher led to the complete block of their stimulatory effect. In the EDTA-treated membranes the enzyme activity was reduced markedly, but it was restored significantly by addition of Ca2+. Calmodulin antagonists--chlorpromazine, W-7, and W-5--caused a dose-dependent decrease of the enzyme activity stimulated by 5 microM Ca2+ with IC50 values of 35, 137, and 174 microM, respectively. The AC-stimulating effects of biogenic amines (serotonin and octopamine) were completely retained in the presence of 2.5 and 100 microM Ca2+, whereas effects of peptide hormones (relaxine and EGF) were hardly changed in the presence of 2.5 microM calcium ions, but were markedly inhibited by 100 microM Ca2+. In the EDTA-treated membranes, the AC effects of biogenic amines were reduced, while the effects of peptide hormones were not revealed. On addition of Ca2+, the AC effects of biogenic amines were completely restored, whereas the effects of peptide hormones were not detected or were restored to a non-significant degree. Calmodulin antagonists slightly affected the AC effects of peptide hormones at concentrations efficient in the case of vertebrate AC, but decreased them markedly at higher concentrations. The AC effects of biogenic amines were little sensitive even to high antagonist concentrations. The obtained data show that targets of action of peptide hormones in the infusorian D. anser cell culture are the AC forms whose activity does not D. depends on calcium cations and possibly is regulated by Ca2+/calmodulin, whereas targets of action of biogenic amines are calcium-independent enzyme forms.

β-adrenoceptor blocker treatment and the cardiac β-adrenoceptor-G-protein(s)-adenylyl cyclase system in chronic heart failure

Recent studies showed that chronic beta-adrenoceptor (AR) blocker treatment exerts beneficial effects in patients with chronic heart failure (CHF). In CHF, sympathetic drive to the heart is increased, and this causes pathological changes in cardiac beta-AR-G-protein(s)-adenylyl cyclase system: Cardiac beta-1 AR are decreased, and amount and activity of cardiac G(i)-protein and G-protein-coupled receptor kinase (GRK) are increased resulting in diminished cardiac beta-AR functional responsiveness. One possible mechanism of beneficial effects of beta-AR blockers could be that they prevent adverse effects of increased sympathetic activity and up-regulate cardiac (and vascular) beta-AR density, and by this, enhance beta-AR-mediated effects. Another possibility could be that chronic beta-AR blocker treatment normalizes activity of G(i)-protein and may thereby restore beta-AR functional responsiveness. Moreover, failing human heart exhibits an inverse force-frequency relationship. beta-AR blockers reduce heart rate; this may, therefore, improve force of contraction. One of the strongest stimuli to activate GRK is increased sympathetic activity (as in CHF) via beta-AR stimulation. beta-AR blockers, by blocking beta-AR, can prevent GRK activation and/or can reduce the (previously enhanced) GRK activity, and this might-at least partly-contribute to beneficial effects of beta-AR blockers in CHF treatment. Finally, the "loss-of-function" Arg389Gly beta-1 AR polymorphism seems to determine heart rate and blood pressure responses to beta-1 AR blocker administration: Arg389Arg beta-1 AR subjects exhibit stronger effects than subjects with one or two Gly389 alleles. Thus, it might be predicted that patients homozygous Arg389 beta-1 AR should be good responders, whereas patients homozygous Gly389 beta-1 AR polymorphism should be poor or non-responders.

Cyclic AMP response element-binding protein in post-mortem brain of teenage suicide victims: specific decrease in the prefrontal cortex but not the hippocampus

Abnormalities in both adenylyl cyclase (AC) and phosphoinositide (PI) signalling systems have been observed in the post-mortem brain of suicide victims. Cyclic AMP response element-binding protein (CREB) is a transcription factor that is activated by phosphorylating enzymes such as protein kinase A (PKA) and protein kinase C (PKC), which suggests that both AC and PI signalling systems converge at the level of CREB. CREB is involved in the transcription of many neuronally expressed genes that have been implicated in the pathophysiology of depression and suicide. Since we observed abnormalities of both PKA and PKC in the post-mortem brain of teenage suicide victims, we examined if these abnormalities are also associated with abnormalities of CREB, which is activated by these phosphorylating enzymes. We determined CRE-DNA binding using the gel shift assay, as well as protein expression of CREB using the Western blot technique, and the mRNA expression of CREB using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) technique in the prefrontal cortex (PFC), and hippocampus obtained from 17 teenage suicide victims and 17 matched normal control subjects. We observed that the CRE-DNA binding and the protein expression of CREB were significantly decreased in the PFC of teenage suicide victims compared with controls. There was also a significant decrease in mRNA expression of CREB in the PFC of teenage suicide victims compared with control subjects. However, there were no significant differences in CRE-DNA binding or the protein and mRNA expression of CREB in the hippocampus of teenage suicide victims compared with control subjects. These results suggest that the abnormalities of PKA, and of PKC, observed in teenage suicide victims are also associated with abnormalities of the transcription factor CREB, and that this may also cause alterations of important neuronally expressed genes, and provide further support of the signal transduction of abnormalities in suicide.

26RFa, a novel orexigenic neuropeptide, inhibits insulin secretion in the rat pancreas

26RFa is a novel orexigenic neuropeptide identified as the endogenous ligand of the orphan G protein-coupled receptor GPR103. GPR103 shares sequence identity with the receptors for neuropeptide-Y and galanin, two peptides known to inhibit insulin secretion. We have investigated the effect of 26RFa on insulin and glucagon secretion in the perfused rat pancreas. 26RFa dose-dependently reduced glucose-induced insulin release, inhibited the insulin responses to both arginine and exendin-4 and did not affect glucagon output. The inhibitory effect of 26RFa on exendin-4-induced insulin secretion was not observed in pancreata from pertussis toxin-treated rats, thus suggesting that 26RFa may inhibit insulin secretion, at least in part, via a pertussis toxin-sensitive G i protein coupled to the adenylyl cyclase system.

Inhibition of Na+/H+‐exchanger with sabiporide attenuates the downregulation and uncoupling of the myocardial β‐adrenoceptor system in failing rabbit hearts

Chronic heart failure (HF) is characterized by left ventricular (LV) structural remodeling, impaired function, increased circulating noradrenaline (NA) levels and impaired responsiveness of the myocardial beta-adrenoceptor (betaAR)-adenylyl cyclase (AC) system. In failing hearts, inhibition of the sodium/proton-exchanger (NHE)-1 attenuates LV remodeling and improves LV function. The mechanism(s) involved in these cardioprotective effects remain(s) unclear, but might involve effects on the impaired betaAR-AC system. Therefore, we investigated whether NHE-1 inhibition with sabiporide (SABI; 30 mg kg(-1) day(-1) p.o.) might affect myocardial betaAR density and AC activity in relation to changes in LV end-diastolic diameter (LVEDD) and LV systolic fractional shortening (LVS-FS) after 3 weeks of rapid LV pacing in rabbits. After 3 weeks of rapid LV pacing LVEDD was significantly increased (Shams 17+/-0.2 mm, n=9 vs 3 wksHF 20+/-0.5 mm, n=8; P<0.05) and LVS-FS decreased (Shams 31+/-1%, n=9 vs 3 wksHF 10+/-1%, n=8; P<0.05). SABI treatment significantly improved LV function independent of whether rabbits were treated after 1 week of pacing (3 wksHF+2 wksSABI (n=7): LVEDD 18+/-1 mm; LVS-FS 16+/-4%) or before pacing (3 wksHF+3wksSABI (n=9): LVEDD 18+/-1 mm; LVS-FS 18+/-6%). After 3 weeks of rapid LV pacing, SABI treatment significantly attenuated increases in serum NA content (Shams 0.83+/-0.19, 3 wksHF 2.68+/-0.38, 3 wksHF+2 wksSABI 1.22+/-0.32, 3 wksHF+3wksSABI 1.38+/-0.33 ng ml(-1)). Moreover, betaAR density (Shams 64+/-5, 3 wksHF 38+/-3, 3 wksHF+2 wksSABI 48+/-4, 3 wksHF+3 wksSABI 55+/-3 fmol mg(-1) protein) and responsiveness (isoprenaline-stimulated AC activity. (Shams 57.6+/-4.9, 3 wksHF 36.3+/-6.0, 3 wksHF+2 wksSABI 56.9+/-6.0, 3 wksHF+3 wksSABI 54.5+/-4.8 pmol cyclic AMP mg(-1) protein(-1) min(-1)) were significantly improved in SABI-treated rabbits. From the present data we cannot address whether the improved betaAR-AC system permitted improved LV function and/or whether the improved LV function resulted in less activation of the sympathetic nervous system and by this in a reduced stimulation of the betaAR-AC system. Accordingly, additional studies are needed to fully establish the cause-and-effect relationship between NHE-1 inhibition and the restoration of the myocardial betaAR system.