Biomedical subjects
Publications and source records attributed to P Simonsson.
Cerebrospinal fluid (CSF), urine, platelet and neuroendocrine challenge tests of monoaminergic function give evidence of monoamines, especially serotonin, playing an important role in suicidal behavior. However, additional clinical, social and biochemical factors are necessary to better define suicide-prone psychiatric patients.
Human neuroblastoma cells SH-SY5Y and neuroblastoma-glioma cells NG 108-15 have been used as models for the elucidation of the effects of ethanol on receptor-mediated phospholipase C activity, c-fos mRNA expression and protein kinase C activity. Cells were exposed to ethanol (0-200 mM) for varying periods up to seven days. Agonist stimulated events were obtained in NG 108-15 cells with bradykinin and in SH-SY5Y cells with carbachol. Chronic ethanol exposure reduced the agonist-stimulated formation of inositol 1,4,5-trisphosphate in NG 108-15 cells and in SH-SY5Y cells. 100 mM ethanol for seven days increased the membrane bound and cytosolic forms of protein kinase C activity in SH-SY5Y cells. Carbachol (1 mM) induced a maximal c-fos mRNA response after 40 minutes in SH-SY5Y cells, an effect that could be mimicked through protein kinase C stimulation by phorbol esters.
In this study the effect of different times of exposure to ethanol (1-7 days, 100 mM) on bradykinin and GTP(S)-stimulated activation of phospholipase C in NG 108-15 cells and on the binding of [3H]bradykinin to its receptors was investigated. Ethanol attenuated both agonist and GTP-analogue-induced hydrolysis of phosphoinositides for a period of up to 4 days of treatment, while exerting no effect on binding to bradykinin receptors. However, after 7 days of exposure to ethanol, the agonist-induced activation of phospholipase C was completely resistant to the inhibitory effects of alcohol. This finding correlated to a change in the affinity of the bradykinin receptor population after 7 days of treatment. The results indicate that bradykinin-induced breakdown of phosphatidylinositol 4,5-bisphosphate adapts to the effects of ethanol, after long-term treatment. Possible adaptative changes taking place at the level of the G protein(s), may induce a shift in the affinity of the receptor population and, consequently, serve as a compensatory mechanism to counteract the inhibitory effect of ethanol.
The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.
It has previously been reported that serotonin-stimulated second messenger formation is inhibited in platelets from alcoholics undergoing detoxification. Serotonin-stimulated signal transduction was therefore analysed in platelets from young post-withdrawal alcoholics to elucidate whether the previously reported inhibition is a state or trait marker of alcoholism. No difference between post-withdrawal alcoholics and controls was found with regard to serotonin-stimulated [32P]-phosphatidylinositol 4,5-bisphosphate hydrolysis or [32P]-phosphatidic acid formation. The results indicate that the inhibited serotonin2 receptor function seen in alcoholics undergoing detoxification is a state-dependent rather than a trait-dependent marker.
The present study reports on the development of a model for maintaining constant ethanol concentrations over time in cell culture media. When neuroblastoma x glioma cells (NG 108-15) were grown in ethanol containing media under standard cultivation conditions in the incubator at 37 degrees C, a 90% evaporation was observed after 24 hr. To counteract evaporation, the cell culture dishes were placed inside polystyrene boxes together with an open dish containing an appropriate amount of ethanol. By using such procedure, the decrease in ethanol concentration in the culture media was completely avoided. Cultivating cells in ethanol-free media inside sealed plastic boxes did not change their viability, growth rate, protein and phospholipid composition of the cells or the pH of the media, compared to cultures grown outside the boxes.
Peripheral serotonergic parameters were studied in 17 patients with a history of suicidal behavior and in 17 age- and sex-matched healthy controls. Serotonin2 receptor function in platelets, measured as serotonin-induced [32P]phosphatidylinositol 4,5-bisphosphate hydrolysis, was significantly higher in patients than in age- and sex-matched controls. Increased serotonin2 receptor transduction was not correlated to psychiatric diagnosis, drug treatment or a history of violent suicide attempt. Monoamine oxidase activity was lower in platelets from patients with a history of suicidal behavior than in controls. In addition, whole blood serotonin was analyzed but no significant alterations were found in blood from patients.
Long-term ethanol exposure is known to inhibit bradykinin-stimulated phosphoinositide hydrolysis in cultures of neuroblastoma x glioma 108-15 cells. In the present study, [3H]bradykinin binding, GTP-binding protein function, and phospholipase C activity were assayed in cells grown for 4 days in 100 mM ethanol with the aim of elucidating the molecular target of ethanol on signal transduction coupled to inositol trisphosphate and diacylglycerol formation. Ethanol exposure reduced guanosine 5'-O-(3-thiotriphosphate) [GTP(S)]- and, to a lesser extent, NaF/AlCl3-stimulated phosphoinositide hydrolysis, whereas it had no effect on the enzymatic activity of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C. [3H]Bradykinin binding in the absence of GTP(S) was not influenced by ethanol exposure. However, the reduction in [3H]bradykinin binding seen in control cells after addition of GTP analogue was inhibited in cells grown in ethanol-containing medium. The results indicate that long-term ethanol exposure exerts its effects on receptor-stimulated phosphoinositide hydrolysis primarily at the level of the GTP-binding protein.
The effect of continuous and intermittent ethanol exposure on the phospholipid composition of Neuroblastoma x Glioma (NG 108-15) cell membranes was investigated. The cells were treated with ethanol for three weeks. Continuous ethanol exposure (150 mM) produced an increase (27%) in the amount of phosphatidylcholine, whereas intermittent ethanol treatment (150 mM) induced a 22% reduction of this lipid. Decreases of phosphatidylethanolamine plasmalogen (8.5%), phosphatidylinositol (16%) and phosphatidylserine (24%) were also seen after intermittent exposure. After binge administration, the concentration of total phospholipids was reduced by 17%, whereas continuous exposure produced a 19% increase. Both intermittent and continuous exposure induced a reduction in the total protein content. No changes in phosphatidic acid, sphingomyelin, phosphatidylcholine plasmalogen or phosphatidylethanolamine (diacyl form) were detected with either treatment. The importance of this study is that ethanol, irrespective of amount, can elicit different effects depending on the pattern of administration.
Astroglial cells in primary culture possess receptors with cyclic AMP and inositol phosphates (IP) as second messengers. The beta-receptor agonist, isoproterenol induces an increase in the accumulation of cyclic AMP, the alpha 2-receptor agonist clonidine inhibits the isoproterenol-induced accumulation of cyclic AMP, while the alpha 1-receptor agonist phenylephrine acts only on the inositol phosphate system. 5-Hydroxytryptamine (5-HT) stimulates, the formation of inositol phosphate, while isoproterenol and clonidine per se do not affect the inositol phosphate system. In the present paper the possibility of interactions between the cyclic AMP and the inositol phosphate transduction systems were investigated. In the presence of 10(-5) M 5-HT, in itself ineffective on the formation of cyclic AMP, isoproterenol stimulated the accumulation of cyclic AMP far more than in the absence of 5-HT. The potentiation was blocked by the 5-HT2 receptor antagonist ketanserin. On the other hand, there were no indications for a beta-receptor influence on the 5-HT-induced inositol phosphate formation. Stimulation of the alpha 2-receptor did not induce accumulation of inositol phosphate but significantly potentiated 5-HT2-receptor transduction, as measured by hydrolysis of phosphoinositide and formation of inositol phosphate. Stimulation by 5-HT also increased the formation of inositol phosphate after adrenergic stimulation and this effect was found to be synergistic at certain concentrations of adrenergic agonists. In addition, there was a statistically significant accumulation of cyclic AMP in the presence of both 5-HT and phenylephrine, none of which stimulated cyclic AMP alone. The results suggest specific interactions between the cyclic AMP and inositol phosphate systems on cultured astroglial cells.
The receptor pharmacology of amperozide was investigated with in vitro radioligand binding technique. Amperozide possessed a high affinity to the 5-HT2 receptors (Ki = 16.5 +/- 2.1 nM) and a moderate affinity to alpha 1-adrenergic receptors of rat cerebral cortical membranes (Ki = 172 +/- 14 nM). The affinity of amperozide for striatal and limbic dopamine D2 receptors was low and not significantly different (Ki +/- S.E.M. = 540 +/- 59 nM vs 403 +/- 42 nM; p less than 0.11, n = 4). The affinity for striatal and limbic 5-HT2 receptors was measured as well and found to be very close to the affinity to the cerebral cortical 5-HT2 receptor. The drug affinity for D2 and 5-HT2 receptors seems thus not to be influenced by the location of the receptor moiety. The affinity for several other rat brain receptors such as 5-HT1A, alpha 2-adrenergic, dopamine D1, muscarinic M1 and M2, opiate sigma and beta 2-adrenergic was low. The pseudo-Hill coefficient of the amperozide competition binding curve was consistently higher than one indicating antagonistic and complex interactions with the 5-HT2 receptor or with alpha 1-adrenergic and dopamine D2 receptors. The antagonistic properties of amperozide were investigated by its ability to antagonize the serotonin-induced formation of inositol-1-phosphate in human blood platelets. Amperozide inhibited this 5-HT2 receptor-mediated intracellular response with similar potency as ketanserin. These results suggest that amperozide is a selective 5-HT2 receptor antagonist.
Serotonin-stimulated activation of phospholipase C in primary astroglial cell cultures was studied as a mean of evaluating the effect of acute ethanol exposition on this signal transduction system. The addition of 50-150 mM ethanol prior to stimulation with 10(-5) M serotonin led to a potentiation of the serotonin-induced [3H]-inositol phosphate formation and an increased incorporation of [3H]-inositol into the three phosphoinositides studied. This potentiating effect of ethanol was observed only when ethanol was added together with serotonin. No stimulatory effect of ethanol per se was found. Furthermore, ethanol had no effect on arginine-vasopressin, bradykinin or phenylephrine stimulated inositol lipid metabolism.
The addition of serotonin to preparations of 32P-labelled human platelets resulted in a time- and dose-dependent hydrolysis of [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) and formation of [32P]phosphatidic acid (PA). This response was inhibited by the serotonin2 receptor antagonist ritanserin, indicating that the stimulation was mediated via the serotonin2 receptor. The addition of 50-150 mM of ethanol prior to stimulation with 10(-5) M serotonin resulted in an increased accumulation of [32P]PA, but had no effect on [32P]PIP2. Ethanol stimulated [32P]PA formation at all serotonin concentrations studied (10(-7)-10(-5) M). Furthermore, in the absence of serotonin, ethanol increased basal [32P]PA formation.
In neuroblastoma x glioma hybrid cells (NG 108-15) labelled with [32P]-trisodium phosphate, [3H]-inositol and [14C]-arachidonic acid, bradykinin stimulated the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) while it had no effect on the release of [14C]-arachidonic acid (AA). The effect on PIP2 was time- and dose-dependent with a maximal effect on [3H]-inositol- and [32P]-labelled cells after 10-30 s of stimulation with 10(-6) M bradykinin. However, the hydrolysis of [14C]-AA labelled PIP2 was delayed compared to the effect on [3H]- and [14C]-PIP2 and was not detectable until after 60 s of stimulation. Bradykinin stimulation resulted in an increased formation of [3H]-inositol phosphates (IP) and [32P]- and [14C]-phosphatidic acid (PA) but the time course for PA formation did not follow the time-course for PIP2 hydrolysis. A reduced labelling of [32P]- and [14C]-phosphatidylcholine was also found in stimulated cells suggesting that PA may derive from other sources than PIP2. In conclusion, our results indicate that bradykinin activates phospholipase C, but not phospholipase A2, in NG 108-15 cells.
Acute ethanol exposure stimulated serotonin2 receptor signalling in cultured astrocytes. Pretreatment with the protein kinase C inhibitor H7 significantly increased the ethanol-induced potentiation of [3H]-inositol phosphates accumulation. The increase could be explained by an augmented activation of phospholipase C. The results indicate a role of PKC for the modulation of ethanol effects on cellular signalling.
The accumulation of inositol monophosphate (IP1) was measured after stimulation of 5-hydroxytryptamine2 (5-HT2) receptors on platelets from alcoholics and healthy controls. In controls, 5-HT induced a dose-dependent response with an EC50 = 2 x 10(-6) M and a maximal response at 10(-5) M. Ritanserin, a selective 5-HT2 antagonist, markedly reduced the accumulation. The IP1 formation after stimulation by 10(-5) M 5-HT was significantly impaired in platelets from alcoholics as compared to controls. This study indicates that the 5-HT2 receptor function is inhibited in alcoholics. It also illustrates the possibility of using IP1 formation in peripheral cells as a mean of studying receptor function in disease.
5-Hydroxytryptamine (5-HT) stimulated the turnover of phosphoinositide in primary cultures of astroglia from the cerebral cortex, striatum, hippocampus and brain stem. Ketanserin and ritanserin, selective antagonists for the central 5-HT2 receptor, inhibited the 5-HT-stimulated formation of inositol monophosphate. In contrast, there was no statistically significant accumulation of cyclic AMP after incubation with different concentrations of 5-HT in any of the cultures studied. The results indicate that astrocytes from various regions of the brain possess 5-HT2 receptors coupled to the formation of inositol phosphates.
Studies on ethanol-exposed animals have revealed changes in anionic phospholipids in brain membranes. The intention of this study was to investigate whether there was a similar effect on man. Assuming platelets to be an adequate model for CNS synaptosomes, concentration and fatty acid composition of anionic phospholipids, phosphatidylserine (PS) and phosphatidylositol (PI) in the platelet membrane from alcoholics after a debauche period were examined and compared to controls. Ethanol effects on neutral lipids were also analysed in order to obtain a comprehensive view. No quantitative difference was found in anionic phospholipids between alcoholics and controls. Fatty acid composition of individual phospholipids revealed significant changes which were more obvious in neutral phospholipids than in anionic. Oleic acid was increased and linoleic and arachidonic acids were decreased. After 1 week of detoxification, the abnormalities did not decrease, on the contrary they increased and total phospholipid concentration per platelet was significantly higher than in controls. It is concluded that the ethanol toxicity on bone marrow hampers the use of platelets as a model for synaptosomes but that the observed lipid abnormalities might play a major role in the impairment of platelet function in alcoholics.