Biomedical subjects

M R Hirvonen

Publications and source records attributed to M R Hirvonen.

At least 19 recordsLinked to original sources

PubMed2001

Inflammatory responses in mice after intratracheal instillation of spores of Streptomyces californicus isolated from indoor air of a moldy building.

Microbial growth in buildings is associated with respiratory symptoms in the occupants. However, the specific effects of the microbes and the way they provoke clinical manifestations are poorly understood. In the current study, mice were exposed via intratracheal instillation to single doses of the spores of Streptomyces californicus, isolated from indoor air of a moisture-damaged building (2.2 x 10(7), 1.1 x 10(8), and 3.3 x 10(8) spores), or lipopolysaccharide (50 microg). Inflammation and toxicity in lungs were evaluated 24 h later. The time course of the effects was explored with the dose of 1.1 x 10(8) spores for up to 7 days. The microbial spores elevated proinflammatory cytokine (i.e., TNFalpha and IL-6) levels in bronchoalveolar lavage fluid (BALF) and in serum in a dose- and time-dependent manner and evoked expression of inducible nitric oxide synthase in BAL cells. Both TNFalpha and IL-6 responses peaked at 6 h after instillation, but TNFalpha leveled off more quickly than IL-6. The cytokine surge was followed by inflammatory cell recruitment into airways. Moreover, the spores increased dose- and time-dependently total protein, albumin, hemoglobin, and lactate dehydrogenase concentrations in BALF during the first 24 h. Histopathological examination of lungs confirmed the inflammatory changes. With the exception of macrophage and lymphocyte numbers, all parameters returned to control level at 7 days. In summary, these observations indicate that the spores of S. californicus are capable of provoking an acute inflammation in mouse lungs and can cause cytotoxicity. Thus, S. californicus can be considered as a species with potential to cause adverse health effects in occupants of moisture-damaged buildings.

Air Microbiology↗

PubMed2001

Differences in inflammatory responses and cytotoxicity in RAW264.7 macrophages induced by Streptomyces Anulatus grown on different building materials.

Streptomyces anulatus, an indicator microbe of mold in buildings, was grown on different building materials in order to study the impact of growth conditions on the ability of the spores of this microbe to induce toxicity and inflammatory responses. The microbes were grown for 2 months on sterilized and unsterilized wood, chipboard, concrete, plaster board and mineral wool in tight glass vessels under humid conditions. The highest microbial spore concentration was detected on the sterilized mineral wool followed by the sterilized plaster board and the unsterilized mineral wool. Mouse RAW264.7 macrophages were exposed in vitro for 24 h to the spores of S. anulatus and the production of the inflammatory mediators, nitric oxide (NO), tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-10 (IL-10) and cytotoxicity, were measured. The dose equivalent to 5 x 10(5) spores/ml of medium was used to compare the different materials. The most intense production of NO (11.6 microM), TNF alpha (560 pg/ml) and IL-6 (2800 pg/ml) in macrophages was induced by the spores grown on sterilized plaster board. They also caused the greatest loss of cell viability (39%). The spores grown on sterilized concrete induced significant production of NO (1.5 microM) and decreased cell viability (22%), and the spores grown on unsterilized and sterilized mineral wool increased production of NO (4.1 microM and 0.8 microM, respectively). The spores did not stimulate production of the anti-inflammatory cytokine IL-10. These results indicate that the ability of S. anulatus to induce inflammatory responses and cytotoxicity in macrophages is dependent on the growth conditions provided by different building materials.

Air Pollution, Indoor↗

PubMed2001

Effect of growth medium on potential of Streptomyces Anulatus spores to induce inflammatory responses and cytotoxicity in RAW264.7 macrophages.

Epidemiological studies have shown an association between microbial growth in buildings and increased risk of respiratory symptoms and disease related to inflammatory reactions in the inhabitants96. The current study examined the affects of growth conditions of Streptomyces anulatus, isolated from indoor air of a moldy building, on the inflammatory potential of spores of this microbe. Spores were harvested from 15 growth media formulations, applied to RAW264.7 macrophages (10(5), 10(6), or 10(7) spores/million cells), and evaluated for the ability to stimulate production of inflammatory mediators and cytotoxicity in these cells 24 h after exposure. Streptomyces anulatus spores induced dose-dependent production of nitric oxide (NO) in macrophages, reaching a level from 4.2 microM to 39.2 microM depending on the composition of the growth medium of the microbe. Expression of inducible NO synthase (iNOS) was detected in macrophages after exposure to spores collected from all growth media. Production of reactive oxygen species (ROS) was significantly increased only by the highest dose of S. anulatus spores grown on glycerol-arginine agar. Furthermore production of cytokines was affected by growth medium; the highest dose-dependent levels of interleukin 6 (IL-6) ranged from 900 to 7800 pg/ml, and the levels of tumor necrosis factor alpha (TNFalpha) varied from 490 to 3200 pg/ml. The amount of dead macrophages after the exposure varied from 11% to 96%, depending also on the growth media of the microbe. Altogether, our results suggest that the growth medium of S. anulatus has a fundamental role in the ability of the spores to induce inflammatory responses and cytotoxicity in mammalian cells.

Air Microbiology↗

PubMed2001

Induction of cytotoxicity and production of inflammatory mediators in raw264.7 macrophages by spores grown on six different plasterboards.

Dampness and microbial growth in buildings are associated with respiratory symptoms in the occupants, but details of the phenomenon are not sufficiently understood. The current study examined the effects of growth conditions provided by six plasterboards on cytotoxicity and inflammatory potential of the spores of Streptomyces californicus, Penicillium spinulosum, Aspergillus versicolor, and Stachybotrys chartarum. The microbes were isolated from mold problem buildings and thereafter grown on six different plasterboards. The spores were harvested, applied to RAW264.7 macrophages (10(4), 10(5), 10(6) spores/10(6) cells), and evaluated 24 h after exposure for the ability to cause cytotoxicity and to stimulate production of nitric oxide (NO), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6). The data indicate clear differences between spores of different microbes in their ability to induce the production of these inflammatory mediators and to cause cell death in macrophages. Also, for each microbe, the induction ability specifically depended on the brand of plasterboard. The spores of Streptomyces californicus collected from all plasterboards were the most potent at inducing NO and cytokine production. Cytotoxicity caused by P. spinulosum and Streptomyces californicus spores was consistent with NO, IL-1beta and IL-6 production induced by those microbes. However, the production of these inflammatory mediators by the spores of Stachybotrys chartarum was not parallel to their ability to cause cell death. The low productions of NO and cytokines were associated with high cytotoxicity caused by the spores of the A. versicolor. These data suggest that growth condition of microbes on different plasterboards affect the ability of microbial spores to induce inflammatory responses and cytotoxicity in macrophages.

Aspergillus↗

PubMed2001

Comparison of mycobacteria-induced cytotoxicity and inflammatory responses in human and mouse cell lines.

Environmental mycobacteria, which are ubiquitous in nature, are also detected in moisture-damaged buildings. Their potential role inducing the adverse health effects associated with living in moisture damaged buildings requires clarification. To establish a model for these studies, we evaluated inflammatory responsiveness in different cell lines exposed to environmental mycobacterial species. Four mycobacterial isolates belonging to Mycobacterium avium complex and Mycobacterium terrae, recovered from the indoor air sampled when a moldy building was being demolished, were studied for their cytotoxicity and ability to stimulate the production of inflammatory mediators in mouse RAW264.7 and human 28SC macrophage cell lines, and human A549 lung epithelial cell line. Lipopolysaccharide (LPS) was used as a positive control. Production of cytokines (tumor necrosis factor alpha, TNF-alpha; interleukin 6, IL-6; and interleukin beta, IL-1beta) was analyzed immunochemically, nitric oxide (NO) by the Griess method, expression of inducible NO synthase with Western blot analysis, and cytotoxicity with the MTT test. Both human and mouse cells produced NO and IL-6 after mycobacterial exposure. Mouse macrophages also showed production of TNF-alpha induced by both mycobacteria and LPS, whereas the human cell lines failed to produce TNF-alpha after mycobacterial exposure and the human epithelial cell line also failed to respond to LPS. Similarly, only mouse macrophages produced IL-1beta. Mycobacterial exposure was not cytotoxic to human cells and was only slightly cytotoxic to mouse macrophages. The results indicate that environmental mycobacterial isolates from moldy buildings are capable of activating inflammatory mechanisms in both human and murine cells. The human and mouse cell lines, however, differ significantly in the grade and type of the responses.

Air Pollution, Indoor↗

PubMed2001

Inflammatory mediators in nasal lavage, induced sputum and serum of employees with rheumatic and respiratory disorders.

Exposure to microbes present in mould-damaged buildings has been linked to increased frequency of various inflammatory diseases. The current study examined differences in inflammatory mediators in nasal lavage (NAL), induced sputum (IS) and serum of occupants with rheumatic or respiratory disorders and their controls, all working in the same moisture-damaged building. Exhaled nitric oxide (NO) measurements, lung function tests, skin-prick tests and health data collection by questionnaire were performed. Concentrations of NO, interleukin (IL)-1, IL-4, IL-6 and tumour necrosis factor-alpha in NAL, IS and serum (excluding NO and IL-1) of the subjects were measured during an occupational exposure period and the vacation period without such exposure. The concentrations of IL-4 in NAL fluid were significantly higher among all occupants during the working period (geometric mean 8.5 microg x mL(-1), range 0-206.5 microg x mL(-1)), as compared to that during vacation (0.4 microng x mL(-1) range 0-3.7 pg x mL(-1)) (p = 0.008). Absence from the work environment also significantly diminished reporting of symptoms. IL-4 levels in the serum of case subjects were significantly higher than in controls. Moreover, employees with respiratory symptoms had markedly higher exhaled NO values than their controls (p = 0.028). In summary, these data suggest that mediators in nasal lavage samples reflect the occupational exposure to moulds, whereas possible indicators of existing disorders are detectable in serum.

Adult↗

PubMed2001

Changes in pro-inflammatory cytokines in association with exposure to moisture-damaged building microbes.

Several epidemiological studies have described an association between adverse health effects and exposure to mould and microbes present in the indoor air of moisture-damaged buildings. However, the biochemical linkage between microbial exposure and the large variety of reported respiratory symptoms is poorly understood. In the present study, the authors compared the respiratory symptoms, the production of inflammatory mediators interleukin (IL)-1, IL-4, IL-6, tumour necrosis factor-alpha (TNF-alpha) and cell count in nasal lavage fluid and induced sputum samples of subjects working in moisture-damaged and control school buildings. The sampling was performed and the questionnaires were completed at the end of the spring term, at the end of the summer vacation (2.5 months), during the winter term and after a 1-week winter holiday. The authors found a significant elevation of IL-1, TNF-alpha and IL-6 in nasal lavage fluid and IL-6 in induced sputum during the spring term in the subjects from the moisture-damaged school building compared to the subjects from the control building. The exposed workers reported sore throat, phlegm, eye irritation, rhinitis, nasal obstruction and cough in parallel with these findings. The present data suggests an association between microbial exposure, and symptoms as well as changes in pro-inflammatory mediators detected from both the upper and lower airways.

Adult↗

PubMed2000

Reproducibility of measurements of exhaled NO, and cell count and cytokine concentrations in induced sputum.

Sputum induction is a noninvasive, well-tolerated method for studying airway inflammation. When induction with hypertonic saline is repeated at short time-intervals (<24 h), the cell profile of sputum has not been reproducible. To determine the proper interval between sampling cell profiles and cytokine contents of sputum samples that had been induced 48 h apart, were compared. In addition, the inducible nitric oxide synthase (iNOS) expression of sputum cells was compared to the levels of exhaled nitric oxide (NO). Sputum induction and measurement of exhaled NO was performed in 31 healthy nonatopic volunteers. Cell differentials were counted. Concentrations of interleukin (IL)-4, IL-6, tumour necrosis factor (TNF)alpha, eosinophil cationic protein (ECP) were measured in sputum supernatant, and iNOS was determined. Reproducibility of cell counts was high (r=0.836 total cells, r=0.762 neutrophils, r=0.966 eosinophils, r=0.742 macrophages). IL-4 (r=0.398), IL-6 (r=0.566), TNFalpha (r=0.658) and ECP (r=0.501) were also less reproducible in healthy volunteers. Consistent with the low levels of NO in the exhaled air (18.5+/-2.6 ppb and 19.3+/-2.8 parts per billion (ppb) on the two study days, r=0.976, p=0.0000), expression of iNOS was not detected. In conclusion, in healthy subjects, induced sputum cell counts are reproducible. Even though the success rate in nonatopic populations is relatively low, sputum induction appears to be a valid method for detecting inflammatory changes within the airways, when being performed 48 h apart.

Administration, Inhalation↗

PubMed1999

Nitric oxide and proinflammatory cytokines in nasal lavage fluid associated with symptoms and exposure to moldy building microbes.

Epidemiological data indicate that living or working in a moldy building is associated with increased risk of respiratory symptoms and disease related to inflammatory reactions, but biochemical evidence linking cause and effect is still scarce. The staff working in a mold-contaminated school, and a reference group without such exposure, were studied. Nasal lavage was performed and health data were collected with a questionnaire at the end of the spring term, after a 2.5-mo summer vacation, and at the end of the fall term. Here we show that concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in nasal lavage fluid were significantly higher in the exposed than in the control subjects at the end of the first exposure period. These inflammatory mediators decreased to reference group concentrations during the period when there was no exposure and the production of NO and IL-6 increased again during the reexposure in the fall term. Reports of cough, phlegm, rhinitis, eye irritation, and fatigue paralleled the changes in the measured inflammatory markers. These results point to an association between inflammatory markers in the nasal lavage fluid, the high prevalence of respiratory symptoms among the occupants, and chronic exposure to molds in the indoor environment.

Adult↗

PubMed1999

Production of reactive oxygen species by man-made vitreous fibres in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) or erythrocytes, isolated from human blood, were exposed to graded doses of asbestos (chrysotile), quartz, or man-made vitreous fibres (MMVF), i.e. refractory ceramic fibres (RCF), glasswool, or rockwool fibres. None of the MMVF affected either the viability of PMNL, as measured by trypan blue exclusion test, or induced haemolysis, whereas the positive controls, quartz and chrysotile, dose-dependently induced haemolysis in PMNL. MMVF did not increase the release of lactate dehydrogenase (LDH) from the PMNL, whereas the positive controls, chrysotile and quartz, induced a marked and dose-dependent release of LDH. When PMNL were exposed to MMVF, some of the fibre types slightly increased the levels of free intracellular calcium ([Ca2+]i) within the cells in a manner similar to that induced by chrysotile or quartz. All MMVF induced a dose-dependent production of reactive oxygen species (ROS) in PMNL, with RCF-induced production of ROS being the most marked. Production of ROS by MMVF seemed to depend on the availability of extracellular calcium because it could be attenuated with a Ca2+ channel blocker, verapamil, or a Ca2+ chelating agent, EGTA. Production of ROS may be a common pathway through which PMNL respond to MMVF-induced cell activation, but alterations of levels of free intracellular Ca2+ do not seem to be an absolute prerequisite for this effect. Fibre length seemed not to be an important factor in affecting the ability of MMVF to induce ROS production in PMNL. However, the balance between different elements in the fibre seemed importantly to affect the biological activity of a fibre.

Adult↗

PubMed1997

Effect of viability of actinomycete spores on their ability to stimulate production of nitric oxide and reactive oxygen species in RAW264.7 macrophages.

Spores of actinomycetes, mesophilic gram-positive bacteria, isolated from moldy houses, induced the expression of inducible NO-synthase (iNOS) with a subsequent NO-production in RAW264.7 macrophages. No differences were detected between production of nitric oxide (NO) by alive or irradiated spores of different strains of Actinomycetes sp. or Streptomyces sp. Moreover, a significant production of reactive oxygen species (ROS) occurred in the macrophages after their stimulation both by alive and irradiation killed spores of actinomycetes. However, ROS-responses in macrophage induced by dead spores were significantly lower compared to those induced by alive spores. The cytotoxicity of the spores of different actinomycetes differed widely. The production of NO and ROS did not depend directly on the viability of the spores, suggesting an important role for cell wall components in the activation of the cells.

Actinomycetaceae↗

PubMed1997

Superoxide formation and macrophage resistance to nitric oxide-mediated apoptosis.

RAW 264.7 macrophages, when challenged with a combination of lipopolysaccharide (10 microg/ml) and interferon-gamma (100 units/ml), respond with endogenous NO. formation, which ultimately results in apoptotic cell death. Apoptosis is detected morphologically by chromatin condensation. Concomitantly we noticed the accumulation of the tumor suppressor protein p53. NO.-derived apoptosis was blocked by the NO.-synthase inhibitor NG-monomethyl-L-arginine. Repetitive treatment of RAW 264.7 macrophages with lipopolysaccharide/interferon-gamma, followed by subculturing viable cells, allowed us to select resistant macrophages which we called RES. RES cells still produced comparable amounts of nitrite/nitrate in response to agonist treatment but showed no apoptotic markers, i.e. chromatin condensation or p53 accumulation. However, RES macrophages undergo apoptosis in the presence of exogenously supplied NO., released from the NO-donors S-nitrosoglutathione or spermine-NO. Assessment of cytochrome c reduction established that RES cells released twice the amount of superoxide compared to RAW 264.7 macrophages under both resting and stimulated conditions. We linked increased superoxide production to cellular macrophage resistance by demonstrating decreased apoptosis after simultaneous application of S-nitrosoglutathione or spermine-NO and the redox cycler 2,3-dimethoxy-1,4-naphthoquinone. Our results suggest that macrophage resistance toward NO.-mediated apoptosis is, at least in part, due to increased superoxide formation. Therefore, the balance between reactive nitrogen and reactive oxygen species regulates RAW 264.7 macrophage apoptosis.

Animals↗

PubMed1997

Species differences in NO formation by rat and hamster alveolar macrophages in vitro.

Nitric oxide (NO) is a cellular mediator and regulator of multiple biologic functions. NO released by alveolar macrophages (AM) is suggested to play a role in mediating pulmonary injury. In murine and rat macrophages, the expression of inducible NO synthase (iNOS) and the release of NO are well established. However, the existence of such a pathway in other species remains controversial. In this study, we examined NO production and iNOS expression by AM from rats and hamsters, two laboratory animal species that are characterized by their disparate pulmonary responses to various inhaled irritants/toxicants. AM were treated with lipopolysaccharide (LPS), interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha) in vitro, and nitrite, the stable oxidation product of NO, was assayed by the Griess reaction. Rat AM produced NO in a dose- and time-dependent manner upon stimulation with LPS and/or IFN-gamma, but not with TNF-alpha. Surprisingly, hamster AM did not release detectable levels of NO after the same treatment. Although iNOS expression was demonstrated in rat AM by immunocytochemical and Western blot analyses, no induction of iNOS expression could be found in hamster AM. Using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, we found that rat and hamster AM could be induced to express iNOS mRNA after treatment with LPS and IFN-gamma. The results presented here indicate that hamster AM, in contrast to rat AM, lack the ability to express iNOS protein and to generate NO in response to LPS, IFN-gamma, or TNF-alpha in vitro. In conclusion, our data suggest striking differences in iNOS regulation and NO production by AM from rats and hamsters, two rodent species that are commonly used in biomedical research and well-known for their disparate responses to pulmonary irritants/toxicants.

Animals↗

PubMed1996

Heat shock proteins and macrophage resistance to the toxic effects of nitric oxide.

Nitric oxide (NO) functions as a pathophysiological mediator in mammalian tissues. Activated macrophages produce NO as a non-specific immune response directed against invading bacteria or micro-organisms. The same macrophages that initiate the production of NO also can be toxically affected by NO. Incubation of RAW 264.7 macrophages with lipopolysaccharide (LPS) and/or interferon-gamma (INF-gamma) induced the formation of NO by the activation of a cytokine-inducible NO synthase (NOS). The viability of these macrophages was inversely correlated with the formation of nitrite, a final NO-oxidation product measurable in the incubation medium. The addition of an NOS inhibitor, NG-monomethyl-L-arginine, diminished NO formation and preserved cell viability in a dose- and time-dependent fashion. Treatment of macrophages with ten cycles of non-lethal doses of LPS and INF-gamma, each followed by subculturing of the surviving cells, resulted in cell resistance to the NO toxic insult induced by LPS and INF-gamma. These resistant macrophages showed a 2-fold increase in the expression of the constitutive heat shock protein (HSC 70) which is known to be involved in protecting cells against the action of various metabolic insults. Our results establish a link between cell resistance to the toxic effects of NO, and the expression of heat shock proteins in RAW 264.7 macrophages.

Animals↗

PubMed1996

Different effects of three bisphosphonates on nitric oxide production by RAW 264 macrophage-like cells in vitro.

The macrophage-suppressive properties of three bisphosphonates were evaluated by studying their effect on nitric oxide (NO) production by activated RAW 264 macrophage-like cells. The cells were activated with 10 micrograms/ml of lipopolysaccharide, and NO was determined as nitrite in the cell culture supernatant. The effect of the drugs on inducible NO synthase was determined by Western blot analysis. As free drugs, clodronate and pamidronate inhibited NO secretion in a dose-dependent manner, whereas alendronate had no effect. Liposome encapsulation enhanced the effect of clodronate by a factor of 7, but the potency of pamidronate weakened slightly when encapsulated in liposomes. The inducible NO synthase expression inside the cells was also decreased by liposomal clodronate. In contrast to pamidronate, clodronate could affect the NO secretion when given to the cells simultaneously with lipopolysaccharide, and the inhibitory action was still seen when the drug was added 2 h after lipopolysaccharide induction. The viability of the cells was not affected by free or liposomal clodronate, whereas pamidronate showed considerable cytotoxicity. This study shows the different actions of these three bisphosphonates on NO production by macrophages and suggests that liposomal clodronate is the most promising bisphosphonate as an anti-inflammatory agent, whereas aminobisphosphonates do not possess anti-inflammatory properties.

Alendronate↗

PubMed1995

The effect of free gallium and gallium in liposomes on cytokine and nitric oxide secretion from macrophage-like cells in vitro.

The aim of this study was to evaluate the effect of gallium nitrate, gallium-nitrilotriacetate (NTA) complex, and liposomal gallium-NTA on IL-6, TNF alpha, and nitric oxide (NO) release from activated macrophages. In addition, the expression of the inducible nitric oxide synthase (iNOS) was determined. Gallium inhibited dose-dependently the secretion of IL-6, TNF alpha, and NO from the LPS-induced macrophage-like RAW 264 cells. Encapsulation of gallium in negatively charged DSPG-liposomes increased its potency 10-50 times and 7-11 times compared to free gallium nitrate and gallium-NTA, respectively. Neither non-loaded liposomes nor NTA alone inhibited cytokine or NO secretion, demonstrating that the observed effects originated from gallium. Liposomal gallium-NTA inhibited the expression of iNOS by the macrophages, while other formulations of gallium had no effect. Thus, gallium, when delivered properly, suppresses macrophage functions by inhibiting the release of inflammatory mediators from the cells.

Animals↗

PubMed1994

TCDD decreases brain inositol concentrations in the rat.

A single dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduced significantly brain regional inositol levels in both the most TCDD-susceptible (Long-Evans; LD50 9.8 micrograms/kg) and the most TCDD-resistant (Han/Wistar; LD50 > 7200 micrograms/kg) rat strain. The decrease emerged earlier in Long-Evans rats but was similar in magnitude at 8 days in both strains. There were some inconsistent and largely dose-independent changes in inositol-1- and inositol-4-monophosphate concentrations at 2 days. On day 8, a tendency towards reduced levels was seen especially in H/W rats. We conclude that TCDD reduces brain inositol levels presumably by inhibiting its synthesis by way of substrate deficit (hypoglycemia), but this effect does not appear to be causally related to the lethal action of TCDD.

Analysis of Variance↗

PubMed1994

Phosphoinositide second messengers in cholinergic excitotoxicity.

Acetylcholine (ACh) is a powerful excitotoxic neurotransmitter in the brain. By stimulating Ca(2+)-mobilizing receptors, ACh, through G-protein(s), stimulates phospholipase C and causes the hydrolysis of a membrane phospholipid, phosphatidylinositol-4,5-bisphosphate to two second messengers, inositol-1,4,5-trisphosphate (ins-(1,4,5)-P3), and diacylglycerol. Ins-(1,4,5)-P3 is important in cholinergic neuronal stimulation, and injury. Cholinergic agonists cause tonic-clonic convulsions which may be either transient or persistent. Even short-term cholinergic convulsions may be associated with neuronal injury, especially in the basal forebrain and the hippocampus. Cholinergic-induced convulsions also elevate levels of brain Ca2+ which precede neuronal injury. Female sex and senescence increase the sensitivity of rats to cholinergic excitotoxicity. Even if cholinergic-induced brain phosphoinositide signalling is likely to trigger cholinergic excitotoxicity, several other processes may be involved in the ensuing neuronal injury. Once initiated, cholinergic convulsions cannot be stopped with cholinergic antagonists such as atropine even though they are effective when given prior to a cholinergic agonist. However, glutaminergic antagonists, and GABAergic agonists, are effective in the attenuation of ongoing cholinergic status epilepticus. Cholinergic brain stimulation may be, in fact, under a partial control of brain GABAergic tonus, but also cause the release of glutamate. Glutamate stimulates inositol lipid signalling in several neuronal cells and, therefore, underlines the significance of inositol lipid signalling in cholinergic-induced excitotoxicity. Moreover, the anatomical distribution of cholinergic brain damage correlates well with that of glutaminergic neurons. Furthermore, glutamate increases neuronal oxidative stress, i.e. it increases the levels of free intracellular calcium, the production of reactive oxygen species, and causes the depletion of neuronal glutathione. The role of excitatory amino acids as common mediators of cholinergic excitotoxicity may offer new insights into the neurotoxic consequences of cholinergic neuronal stimulation.

Acetylcholine↗