AIM:Findings of hypoxia prior to death and involvement of a dysregulation of the serotonergic network in sudden infant death syndrome (SIDS) may indicate that brain-derived neutrophic factor (BDNF) also is of importance with regard to sudden unexpected infant death. Based on this, the purpose of this study was to investigate the BDNF val66met polymorphism in SIDS cases, cases of infectious death and controls.METHODS:The polymorphism was investigated in 163 SIDS cases, 34 cases of infectious death and 121 controls, using real-time PCR and fluorescence melting curve analysis.RESULTS:There were no differences in val66met genotype distribution between neither the SIDS cases nor the cases of infectious death and controls (p = 0.95 and p = 0.52, respectively).CONCLUSION:The study indicates that the val66met polymorphism is not important for sudden unexpected infant death. However, several other SNPs in the BDNF gene, as well as in other genes involved in this pathway, including G-protein, have to be investigated to fully exclude any involvement of BDNF in SIDS.

PURPOSE:Experimental studies suggest a role of G protein-mediated signaling pathways in epileptogenesis. A genetic variation affecting the G protein subunit Gbeta3 denoted the C825T polymorphism has been reported to increase the signaling efficiency through G(i) proteins and to modify responses to certain drugs. The C825T polymorphism has also been associated with several diseases including hypertension, diabetes type II, obesity, and major depressive disorder. In this study, we have explored whether the G protein polymorphism C825T is associated with or influences temporal lobe epilepsy (TLE).METHODS:The study included 227 TLE patients, 186 controls, and 106 family members of TLE patients. DNA was extracted from blood samples and typing of the polymorphism was performed. Case record forms were analyzed for all the homozygote TLE patients and homozygote controls, i.e., carrying the TT genotype as well as for 28 matched TLE patients (16 females, 12 males) without the polymorphism (CC genotype).RESULTS:Typing of the C825T polymorphism showed that 6.0% of the TLE patients, 7.0% of the controls, and 7.5% of the family members were homozygote for the polymorphism; i.e., carrying the TT genotype. TLE patients carrying the TT genotype had higher severity score on eight out of nine predefined parameters compared with the TLE patients without polymorphism, i.e., carrying CC genotype. TT genotype TLE patients also had increased body mass index, body weight, and waist circumference compared with the TLE patients carrying the CC genotype. There was no increased frequency of hypertension or diabetes.CONCLUSIONS:There was no increased frequency of TLE between the carriers of the TT genotype compared with the healthy controls and/or family members without epilepsy. However, the TLE patients with the TT genotype showed tendencies of a more severe disease phenotype.

Sodium nitroprusside (SNP), a nitric oxide (NO.) donor, stimulates glucose uptake in skeletal muscle. We investigated the stimulatory effect of SNP on glucose uptake in cardiomyocytes and the possible role of soluble guanylate cyclase, phosphatidylinositol-3-kinase (PI-3-kinase) and the mitogen-activated protein kinases (MAPKs). Cardiomyocytes were isolated from adult male Wistar rats by trypsin/collagenase perfusion and glucose uptake determined from the accumulation of 3H-2-deoxyglucose. SNP caused a dose-dependent increase in glucose uptake with 200-300% increase at 30 mM. Cytochalasin B completely prevented the SNP-induced increase in glucose uptake. 8-Br-cGMP (100 microM) and the NO. donor spermineNONOate (100 microM) were without effect on basal glucose uptake. SNP-stimulated glucose uptake was not inhibited by the guanylate cyclase inhibitor ODQ (10 microM). Sodium ferrocyanide (Na4Fe(CN)6), a compound structurally related to SNP, but without any NO. group, also stimulated glucose uptake in cardiomyocytes suggesting that the effect of SNP could be unrelated to liberation of NO. Wortmannin, an inhibitor of PI-3-kinase, inhibited insulin-stimulated glucose uptake completely but did not affect SNP-stimulated glucose uptake. SNP-stimulated glucose uptake was inhibited by 50 microM PD 098059 (inhibitor of the MAPK-kinases that activate external regulated kinase [ERK1/2]) and by 50 microM SB203580 (inhibitor of p38MAPK). In conclusion, high SNP concentrations dose-dependently stimulate glucose uptake in cardiomyocytes and our data suggest a role for MAPK signalling, but not PI-3-kinase and soluble guanylate cyclase, in stimulation of glucose uptake.

It was previously found that pertussis toxin (PTX) pretreatment inhibits the activation of extracellular signal‐regulated kinases ERK1 (p44mapk) and ERK2 (p42mapk) in hepatocytes in response to either agonists that bind to heptahelical receptors or epidermal growth factor (EGF), suggesting a role of Gi proteins in stimulatory mechanisms for ERK1/2. The present work shows that ERK1/2 is activated in a PTX‐sensitive way not only by vasopressin, angiotensin II, prostaglandin (PG) F2α, α1‐adrenergic stimulation, and EGF but also by agents whose actions bypass receptors and stimulate protein kinase C (PKC) and/or elevate intracellular Ca2+, such as 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), exogenous phosphatidylcholine‐specific phospholipase C (PC‐PLC, from Bacillus cereus), thapsigargin, and the Ca2+ ionophore A23187. Under the same conditions, PTX did not affect agonist stimulation of phosphoinositide‐specific phospholipase C (PI‐PLC) (IP3 generation), and did not reduce the activation by these agents of phospholipase D (PLD). The results suggest that in hepatocytes a PTX‐sensitive mechanism, presumably involving Gi proteins, exerts a stimulatory effect on ERK at a level distal to receptor coupling, acting either as an integral part of the signaling pathway(s) or by a permissive, synergistic regulation. J. Cell. Physiol. 184:27–36, 2000. © 2000 Wiley‐Liss, Inc.

The role of diacylglycerol (DAG) in hormonal induction of S phase was investigated in primary cultures of rat hepatocytes. In this model, several agonists that bind to G protein-coupled receptors act as comitogens when added to the cells soon after plating (i.e., in Go/early Gl phase), while the cells are most responsive to the mitogenic effect of epidermal growth factor (EGF) at 24–48 h of culturing (i.e., mid/late Gl). It was found that the cellular concentration of DAG rose markedly and progressively during the first 24 h of culturing. Exposure of the hepatocytes at 3 h to αl-adrenergic stimulation (norepinephrine with timolol), vasopressin, or angiotensin II further increased this rise, producing a sustained increase in the DAG level. Norepinephrine, which was the most efficient comitogen, produced the most prolonged DAG elevation. In contrast, no significant increase of DAG was found in response to EGF, neither at 3 nor at 24 h, using concentrations that markedly stimulated the ERK subgroup of the mitogen-activated protein kinases (MAPK) and DNA synthesis. Addition of Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC) strongly elevated DAG, while Streptomyces phospholipase D (PLD) increased phosphatidic acid (PA) but not DAG. B. cereus PC-PLC and the protein kinase C (PKC) activator tetradecanoyl phorbol-acetate (TPA), like norepinephrine, vasopressin, and angiotensin II, stimulated MAPK and enhanced the stimulatory effect of EGF on DNA synthesis. The PKC inhibitor GF109203X did not diminish the effect of EGF on MAPK or DNA synthesis, but strongly inhibited the effects of norepinephrine, vasopressin, angiotensin II, TPA and B. cereus PC-PLC on MAPK and almost abolished the enhancement by these agents of EGF-stimulated DNA synthesis. These results suggest that although generation of DAG is not a direct downstream response mediating the effects of the EGF receptor in hepatocytes, a sustained elevation of DAG with activation of PKC markedly increases the responsiveness to EGF. Mechanisms involving DAG and PKC seem to play a role in the comitogenic effects of various agents that bind to G protein-coupled receptors and activate the cells early in Gl, such as norepinephrine, angiotensin II, and vasopressin. J. Cell. Physiol. 180:203–214, 1999. © 1999 Wiley-Liss, Inc.

Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F-2 alpha, activated the ERK1 (p44(mapk)) and ERK2 (p42(mapk)) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3-5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a G(q)-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PCF2 alpha. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that G(i)-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor. (C) 1998 Wiley-Liss, Inc.

Several prostaglandins inhibit the cAMP response to glucagon and beta-adrenergic stimulation in hepatocytes. To probe the mechanism of this inhibition, we have examined in primary hepatocyte cultures how pretreatment with pertussis toxin (islet-activating protein) influences the ability of the cells to respond to hormones and prostaglandins. Pertussis toxin augmented the effects of glucagon, epinephrine and isoproterenol, and also markedly enhanced the cAMP response to prostaglandin E1 (PGE1). Furthermore, whereas PGE1, PGE2, PGI2 and PGF2 alpha attenuated the cAMP responses to glucagon in control cultures, this inhibition was abolished in cells pretreated with pertussis toxin. A more detailed comparison was made of the effects of PGE1 and PGF2 alpha. In cells not treated with pertussis toxin, both these prostaglandins at high concentrations reduced the cAMP response to glucagon and isoproterenol by approximately 50%, but dose-effect curves showed that PGE1 was about 100-fold more potent as an inhibitor than PGF2 alpha. Pertussis toxin abolished the inhibitory effects of PGE1 and PGF2 alpha with almost identical time and dose requirements. The results obtained with PGE1, PGE2, PGI2 and PGF2 alpha suggest that prostaglandins of different series attenuate hormone-activable adenylate cyclase in hepatocytes through a common mechanism, dependent on the inhibitory GTP-binding protein.

Adult male rat hepatocytes, which normally respond poorly to β‐adrenergic agents, acquire such responsiveness during primary monolayer culture. We here show that the rise in catecholamine‐sensitive adenylate cyclase activity in hepatocytes in vitro is closely paralleled by an increase in the ability to bind the β‐adrenoceptor ligand [125I]cyanopindolol. The emergence of β‐adrenergic responsiveness did not require cell attachment or serum. Addition of dexamethasone, insulin, thyroxine or dihydortestosterone to the cultures, singly or in combination, did not prevent the augmented β‐adrenergic responsiveness. The increase in catecholamine‐sensitive adenylate cyclase activity and [125I]cyanopindolol binding could be blocked by cycloheximide or actinomycin D. Exposure of the cultures to isoproterenol at 3‐hourly intervals led to a dose‐dependent suppression of the rise in isoproterenol‐responsive adenylate cyclase and prevented the increase in β‐adrenoceptor binding.