Decreased levels of cGMP in vitreous and subretinal fluid from eyes with retinal detachment.

BACKGROUND: Cyclic guanosine monophosphate (cGMP) is produced in different retinal cells, including photoreceptor cells, wherein cGMP mediates photo-transduction. CGMP is degraded by phosphodiesterases (PDE). The aim was to investigate whether retinal detachment alters intraocular cGMP levels in human eyes. METHODS: cGMP and PDE were determined in vitreous fluid from 50 eyes with a retinal detachment (group I) and in 20 control samples (group II) of vitreous fluid from eyes without retinal detachment. Group III consisted of subretinal fluid samples from 70 eyes with retinal detachment. RESULTS: cGMP in vitreous fluid from eyes with retinal detachment (6.5 (SD 1.7) nM) was decreased compared to controls (67.1 (10.0) nM) (p<0.0001). In subretinal fluid, the mean level of cGMP was 2.4 (0.2) nM. No PDE could be detected in any of the intraocular fluid samples of patients nor controls. A decrease in the mean level of cGMP in subretinal fluid of eyes with retinal detachment correlated with a longer duration of detachment (r = -0.45, p = 0.007). CONCLUSIONS: Retinal detachment was found to be associated with a decrease in vitreous cGMP concentration. In subretinal fluid, a low cGMP level correlated inversely with the duration of the detachment.

Phosphoinositide kinase activities in synaptosomes prepared from brains of patients with Alzheimer's disease and controls.

Previously, phosphatidylinositol (PI) kinase activity in cytosolic fractions prepared from postmortem tissue of the cerebral cortex from patients with Alzheimer's disease (AD) appeared to be lower than that of age-matched controls [Jolles et al., J. Neurochem., 58 (1992) 2326-2329]. In the study presented here, PI and PIP (phosphatidylinositol phosphate) kinase activities were studied in synaptosomes prepared from postmortem brain tissue of AD patients and age-matched controls. Firstly, PI kinase activity in synaptosomes prepared from the frontal superior gyrus of AD brain was 30% lower than in synaptosomes prepared from postmortem tissue of control brain. PIP kinase activity was the same in AD and control synaptosomes. Secondly, the yield of synaptosomal protein (micrograms protein per mg tissue wet weight) was lower in preparations from AD brain than in preparations from control brain, which could be a manifestation of a loss of presynaptic terminals in the frontal cortex. These results suggest that the difference in PI kinase activity between AD and control brain tissue may originate from differences in intact neurons in view of the fact that synaptosomes can originate only from intact neurons.

Evidence for a selective decrease in type 1 phosphatidylinositol kinase activity in brains of patients with Alzheimer's disease.

We have previously shown that phosphatidylinositol (PI) kinase activity is 40-50% lower in cytosolic fractions of neocortical regions from brains of patients with Alzheimer's disease (AD) in contrast to phosphatidylinositol phosphate (PIP) kinase activity, which was not affected. After preparing different enzyme fractions by solubilization, the PI kinase activity in the salt-solubilized protein preparation of the temporal cortex of AD brains was predominantly affected (70% decrease). PIP kinase activity in AD brains was not different from that of control brains in any of the fractions tested. PI kinase in the salt-solubilized fractions was inhibited (-75%) by 1% Triton X-100, whereas the PI kinase in the detergent solubilized protein preparation was stimulated (+80%) by 1% Triton X-100. PI kinase activity in the salt-solubilized protein preparation was almost unaffected by adenosine in contrast to PI kinase activity in the detergent solubilized protein preparation, which was strongly inhibited by adenosine. These results indicate that the PI kinase that is specifically affected in AD is the PI 3-kinase, or type 1 PI kinase, because the fraction which was affected most severely has (1) a cytosolic localization; (2) a high sensitivity to inhibition by the non-ionic detergent Triton X-100, and (3) an insensitivity to adenosine inhibition, which are characteristic features of type 1 PI kinase. The relevance of our findings is that type 1 PI kinase is thought to be involved in the regulation of cytoskeletal turnover processes.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of age on phosphatidylinositol kinase, phosphatidylinositol phosphate kinase and diacylglycerol kinase activities in rat brain cortex.

A previous study, in which a lysed fraction was used with endogenous phospholipids as substrate, revealed age-related changes in PA and PIP2 formation but not in PIP formation (Bothmer et al., Neurochem. Int. 21, 223-228, 1992). To rule out the influence of substrate availability in the present study, the effect of age on PI kinase, PIP kinase and DAG kinase activities was studied with exogenous phospholipids as substrate in the cerebral cortex from 8-month-old, 14-month-old and 26-month-old Brown Norway rats. PI kinase activity was predominantly located in a tight membrane-bound protein fraction, DAG kinase activity in cytosolic and loosely membrane-bound protein fractions, and PIP kinase activity was present in all three protein preparations. The effects of age were limited to a small increase in kinase activity in the tight membrane-bound protein fraction in 14-month-old and 26-month-old rats compared to 8-month-old rats, and a 10% decrease in PIP kinase activity in the cytosolic protein fraction in 14-month-old and 26-month-old rats compared to 8-month-old rats. DAG kinase activity showed no age-related changes. In conclusion, one should take care in comparing rat aging with human aging as PI kinase activity shows an age-related decline in human brain cortex (Jolles et al., J. Neurochem. 58, 2326-2329, 1992). Furthermore, previously reported decreases in PA formation rates in rat brain are probably not due to changes in DAG kinase itself but to changes in DAG availability, although further experimental evidence is needed to confirm this conclusion.

Platelet phosphatidylinositol kinase activity is not altered in Alzheimer disease.

We previously reported a specific decline in phosphatidylinositol (PI) kinase activity in the neocortex of patients with Alzheimer disease (AD) as compared to controls, whereas phosphatidylinositol phosphate (PIP) kinase activity appeared not to be affected (Jolles et al., 1992). In search of a possible systemic effect of AD, in the present study we investigated phosphoinositide kinase activity in platelets from patients with AD and from control subjects. The study was based on the notion that disease-specific abnormalities in the brain could be reflected in blood platelets. PI kinase activity was studied in platelet homogenates and in a salt-solubilized protein fraction of platelets, because of the difference in subcellular localization of the different types of PI kinases. In addition, NADH cytochrome-C reductase was measured in platelet homogenates as a marker for the endoplasmic reticulum, to detect a possible proliferation of the endoplasmic reticulum. AD patients and normal elderly controls showed no difference in PI kinase activity in either enzyme fraction. Furthermore, NADH cytochrome-C reductase activity and the protein/phospholipid ratio per 10(6) platelets were the same for AD patients and controls. This was taken as an indication that platelets in AD patients do not show proliferation of intracellular membranes.

Reduced phosphatidylinositol kinase activity in Alzheimer's disease: effects of age and onset.

Phosphatidylinositol kinase (PI kinase) and phosphatidylinositol phosphate kinase (PIP kinase) were assayed in a membrane-free cytosolic fraction prepared from the medial temporal cortex of patients with Alzheimer's disease (AD) and nondemented controls, with exogenous lipids as substrate. 32P-PIP formation appeared to be reduced by 21% in patients with AD who died before 80 years, compared to matched controls. In addition, there was an age-related decrease in PI kinase activity in the control group. In the AD patients there was no age-related decrease. Very old AD patients (older than 80 when they died) with a short duration of the disease were characterized by increased PI kinase activity. No differences were found in the closely related enzyme PIP kinase. The results are indicative for heterogeneity in AD.

Pitfalls in the measurement of plasma osmolality pertinent to research in vasopressin and water metabolism.

The reliability of measurements of plasma osmolality is known to be biased by technical artifacts, such as the anticoagulant and the osmometric technique used; the resulting measurement errors therefore may cause errors in interpretation of data. In assessing the potential biasing influence of procedural variables, we found that the temperature at which fresh plasma samples were stored, the duration of storage, and the freezing and thawing of samples appeared to significantly (P < 0.01) affect osmolality values around the narrow physiological range. These factors should be considered in the interpretation of studies on the osmoregulation of vasopressin secretion. In particular, the results suggest that data obtained for any but fresh samples, whether frozen-thawed samples or samples stored at room temperature, are unreliable.

Age-related changes in concentrations of vasopressin in the central nervous system and plasma of the male Wistar rat.

The results of studies on the influence of age on concentrations of vasopressin (VP) in blood plasma and hypothalamic and extrahypothalamic brain sites have not been unequivocal. Studies on extrahypothalamic concentrations of VP in the aging rat have used two age groups only and have mainly provided semiquantitative data. For these reasons we determined, by radioimmunoassay, the concentrations of vasopressin in thirteen brain structures and in the plasma of 3-, 10-, 20- and 28-month-old male Wistar rats. Age-related decreases in VP concentrations were found in the pituitary gland, hypothalamus, thalamus, midbrain, medulla oblongata, amygdala and pineal gland, while an increase was noted in plasma. Decreases in the concentration of VP in the amygdala and pineal gland occurred between 3 and 10 months of age and probably represent developmental changes. In the pituitary, thalamus, midbrain, medulla oblongata and plasma, differences in the concentration of VP were also found between 10-month-old and older animals and are probably related to aging. The finding of increased plasma VP concentrations in aged animals agrees with the notion that neuronal function does not necessarily decline with age and suggests that neurons may even be activated. Age-related changes in VP concentrations were not observed in the other structures examined. It has been reported that the VP innervation of a number of brain structures depends on testosterone. Despite reports to the contrary VP concentrations do not generally decline in these structures with aging.

Evidence for a new inositol phospholipid in rat brain mitochondria.

Phosphorylation of phosphatidylinositol (PI), phosphatidylinositol monophosphate (PIP) and diacylglycerol (DAG) was studied in rat brain cortex myelin, synaptosomal and mitochondrial fractions, with ATP as phosphate donor and endogenous phospholipids as substrate. All fractions had PI, PIP and DAG phosphorylating activity with their own characteristic subcellular distribution. However, in the mitochondrial fraction an unidentified lipid was phosphorylated, which had a slower Rf value than PIP2 on TLC. After hydrolysis of the polar head group of the lipid and separation on anion exchange columns, it appeared to be a phosphoinositide. The elution profile showed that it was not phosphatidylinositol trisphosphate, or a lyso-compound. The available evidence suggests that the unknown inositol phospholipid in rat brain mitochondria is a phosphatidylinositol 4,5-bisphosphate isomer, although the possibility of it being a glycosyl-phosphoinositide cannot be excluded.

Brain phosphatidic acid and polyphosphoinositide formation in a broken cell preparation: regional distribution and the effect of age.

The effect of age on phosphate incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidic acid (PA) was studied. Lysed crude synaptosomal fractions of different brain regions of 3-month-old and 32-month-old Brown Norway rats were used. The brain regions tested were the hippocampus, frontal cortex, occipital/parietal cortex, entorhinal/pyriformal cortex, striatum/septum, thalamus and hypothalamus. The individual specific phosphorylating activities were unevenly distributed within the brain of Brown Norway rats. Strikingly, the distribution of phosphate incorporation into PIP2 was opposite from that of phosphate incorporation into PA. Phosphate incorporation into PA decreased (-15%) with age in almost all brain regions tested, whereas phosphate incorporation into PIP2 decreased with age only in the frontal cortex (-20%) and in the hypothalamus (-8%). The effects of age may reflect a deterioration of phosphoinositide metabolism, with its function in signal transduction coupled to receptors via G-proteins, in the brain regions involved. In addition, there was an age related decrease in protein content and total phospholipid phosphorus content of lysed crude synaptosomal preparations of all brain regions. The high correlation between the changes in these parameters may be indicative of a decrease in the number or size of synaptosomes with age in the brain regions involved.

Phosphatidylinositol kinase is reduced in Alzheimer's disease.

Phosphatidylinositol (PI) kinase and PI phosphate (PIP) kinase activities were measured in postmortem samples of brain tissue from patients with Alzheimer's disease and nondemented control subjects. A membrane-free cytosolic fraction from four neocortical locations, with exogenous inositol lipids as the substrate, was used. Tissue from patients with Alzheimer's disease was characterized by reduced PIP formation; the reduction was 50% in prefrontal cortex, temporal cortex, and parietal cortex and 40% in precentral gyrus. In contrast, no alterations were found in PI bisphosphate formation in these four neocortical locations. The specific changes in PI kinase but not PIP kinase activity suggest that the findings may have functional relevance to the involvement of brain membrane processes in Alzheimer's disease.

Changes in plasma vasopressin concentration and plasma osmolality in relation to age and time of day in the male Wistar rat.

The influence of age on several parameters related to water balance was studied in Wistar rats. Plasma AVP concentration and plasma osmolality were increased at midday in 21-month-old rats as compared with 3- and 4-month-old rats. Daily water intake per 100 g body weight was reduced in 14- and 21-month-old rats as compared with 3- and 4-month-old rats, but total water intake was unaltered. These results suggest that there is a change in water balance in Wistar rats with age. In order to obtain information about the influence of age on daily fluctuations in plasma AVP concentration and osmolality these parameters were determined in 4-month-old Wistar rats sacrificed at 2 h intervals during the day and in 20- and 31-month-old rats sacrificed at 8 h intervals. Plasma AVP concentrations were low during the light period and high during the dark period in 4-month-old rats. The relationship between plasma osmolality and plasma AVP concentration was dependent on the time of day in 4-month-old-rats. Plasma AVP concentrations were higher at 16.00 than at 08.00 and 24.00 in 20-month-old rats, and higher at 24.00 than at 08.00 and 16.00 in 31-month-old rats. In contrast to the plasma AVP concentration during the light period, the average daily AVP concentration (average of plasma AVP concentrations at 08.00, 16.00 and 24.00) was increased in 31-month-old rats only. The relationship between plasma osmolality and plasma AVP concentration was not age-related.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphatidic acid and polyphosphoinositide formation in a broken cell preparation from rat brain: Effects of different incubation conditions.

The formation of phosphatidic acid (PA) and polyphosphoinositides in rat brains in vitro was studied under hypotonic conditions by incubating a lysed crude synaptosomal fraction with [?-(32)P]ATP for 10 s. Only phosphatidic acid, phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP(2)) were labelled. The formation of PIP(2) decreased with increasing preincubation times. PIP and PA formation, however, were not affected. The rate of phosphorylation of PIP, PIP(2) and PA was linear with incubation time for at least 10 s, indicating that the ATP/enzyme ratio was sufficiently high. The maximal incorporation of phosphate into the three phospholipids was found at 30 degrees C. Ten millimolar Mg(2+) and pH 6.5 were the best compromise for measuring the incorporation of phosphate into PIP, PIP(2) and PA simultaneously. A sodium acetate-magnesium acetate buffer system gave optimal kinase activities. The results obtained show that, with minor adjustments of the incubation conditions, it is possible to measure simultaneously the formation of PIP, PIP(2) and PA.