Phenylmethylsulfonyl fluoride inhibitory effects on acetylcholinesterase of brain and muscle.

Differential inhibition of brain versus peripheral acetylcholinesterase (AChE) by phenylmethylsulfonyl fluoride (PMSF) suggested that PMSF might preferentially inhibit different AChE molecular forms. AChE inhibition was examined after systemic and in vitro PMSF treatment. Systemic administration resulted in no overt behavioral changes but produced a 71% reduction in brain AChE; hemidiaphragm, extensor digitorum longus and soleus muscles showed 65, 50 and 41% reductions. Muscle asymmetric AChE was reduced to the greatest extent (50-80%). The tetrameric form was inhibited in brain and hemidiaphragm (60-76%) but spared in other muscles (18-22%). Monomeric AChE was spared in all tissues. When PMSF was added to a muscle homogenate all forms were inhibited equally. Purified monomer and tetramer forms were inhibited equally in vitro. These results suggest that PMSF inhibition of AChE is a consequence of a selective inhibition of membrane-associated forms and that the apparent brain selectivity is related to the greater fraction of membrane-associated AChE in brain.

Age-related changes in activity of Fischer 344 rat brain acetylcholinesterase molecular forms.

Total acetylcholinesterase (AChE) and the molecular forms of the enzyme from six brain regions were compared in young adult (6 mo) and aged (24 mo) Fischer 344 rats. Total AChE activity was significantly reduced in aged striatum (48.7%), parietal cortex (39%), cerebellum (30.2%), and medulla/pons (23.1%). Forebrain of aged rats showed nonsignificant reduction of AChE (18.4%), but olfactory bulbs exhibited no differences in aged rats. The ratio of G4/G1 molecular forms, as isolated on sucrose density gradients, was unaltered in all aged rat brain tissues examined. These results indicate that aged rats exhibit reduced brain AChE, but there is no evidence for selective effects on individual molecular forms.

Characteristics of the myocardial PM-FABP: effect of diabetes mellitus.

Properties of the myocardial PM-FABP were studied in normal and STZ-diabetic rats. The fluorescent fatty acids trans-parinaric and cis-parinaric acids were used as analogs of straight-chain (saturated) and kinked-chain (unsaturated) fatty acids respectively. Parinaric acid binding was sensitive to trypsin. Trans-parinaric acid binding was more sensitive to this protease than the binding of cis-parinaric acid. Based on the difference in sensitivity of parinaric acid binding we believe that there are two separate binding sites associated with myocardial PM-FABP; one for unsaturated fats and the other for saturated fats. Diabetes enhanced both cis- and trans-parinaric acid binding capacity in cardiomyocytes; cis-parinaric acid by 2 fold and trans-parinaric acid by 2.6 fold. In addition, there was a concomitant accumulation of free fatty acids and triglycerides in the hearts of the diabetic animals. There was a 2.2 fold increase for fatty acids and a 1.6 fold increase for trigylcerides. This association between myocardial fatty acid build-up and enhanced myocardial PM-FABP during diabetes suggest that this carrier protein might have contributed to lipid accumulation in the hearts of the diabetic rats.

Nerve growth factor in Alzheimer's disease: increased levels throughout the brain coupled with declines in nucleus basalis.

The current study analyzed NGF protein levels in the brains of patients with Alzheimer's disease (AD) as compared with aged neurologically normal individuals. An established two-site ELISA was used to measure NGF-like immunoreactivity in the hippocampus, superior temporal gyrus, superior frontal gyrus, inferior parietal lobule, frontal and occipital cortical poles, cerebellum, amygdala, putamen, and nucleus basalis of Meynert (nbM). ChAT activity was assayed in adjacent tissue samples. NGF levels were also evaluated in Parkinson's disease for comparison with both AD and age-matched control cases. Regardless of the brain bank (University of Cincinnati, Rush Presbyterian St. Luke's Medical Center in Chicago, or University of Alabama at Birmingham), NGF-like activity was at least moderately increased with AD in virtually every brain region examined except for the nbM, in which significant declines were observed. NGF levels were also increased when compared with age-matched Parkinson's cases (frontal cortex). NGF-like activity was not related to age at onset or disease duration in AD cases, nor did NGF levels correlate with age at death in the control or AD groups. Correlations between ChAT and NGF-like activity across brains varied considerably and were generally not significant. The present findings indicate that AD is characterized by a widespread increase in cortical and subcortical NGF. Although a correlation with ChAT activity was not observed in cortex, the AD-related decline in NGF found in nbM is consistent with the possibility of impaired retrograde transport of NGF to this region.

Effect of hydralazine on myocardial plasma membrane fatty acid binding protein (PM-FABP) during diabetes mellitus.

Chronic treatment with the antihypertensive drug hydralazine did not affect the hyperglycemic state of streptozotocin (STZ)-diabetic rats but did prevent the serum hyperlipidemia that is synonymous with these diabetic animals. After 6 weeks, untreated STZ-diabetic rats exhibited a 659% increase in serum triglycerides and 292% increase in serum cholesterol versus age-matched non-diabetic rats. Hydralazine-treated STZ-diabetic rats had serum triglyceride and cholesterol levels that did not differ from controls. Myocytes from control rats showed a preference for binding of the unsaturated fatty acid analog cis-parinaric acid vs the saturated fatty acid analog trans-parinaric acid. This preference was not altered in STZ-diabetic rat myocytes; hydralazine-treatment of STZ-diabetic rats also showed no change in fatty acid preference. STZ-diabetes caused a decrease in the affinity (Kd) for the trans, but not the cis-parinaric acid. However, total binding of both analogs was increased in STZ-diabetes. Hydralazine treatment of STZ-diabetic rats resulted in even greater total binding of both analogs. Affinity for the trans analog was further decreased in these hydralazine-treated rats, but the affinity for the cis analog was increased beyond control animals. These results suggest that the diabetic state influences the binding characteristics of the myocardial PM-FABP and that hydralazine, while reducing serum lipids in diabetes, has complex effects on the fatty acid binding by this protein.

Role of exogenous adenosine as a modulator of theophylline toxicity.

OBJECTIVE: In animal models, exogenous adenosine can reverse theophylline-induced neurologic toxicity. Thus, we examined the ability of adenosine to oppose the cardiovascular toxicity of theophylline. DESIGN: Open-label, dose-response trial. SETTING: Animal laboratory within a college of pharmacy. SUBJECTS: Ten chloral hydrate-anesthetized male Sprague-Dawley rats. INTERVENTIONS: Indwelling catheters were surgically inserted into the right carotid artery and right external jugular vein. Subsequently, escalating doses of adenosine (5 to 2000 micrograms) were given at theophylline doses of 0, 10, 20, 30, 40, and 50 mg/kg until a maximum slowing in heart rate occurred. MEASUREMENTS AND MAIN RESULTS: Left ventricular systolic pressure and maximum positive maximal change in pressure over time (+dP/dt) were determined at baseline and at the time of maximum heart slowing, during adenosine, for each theophylline dose (0 to 50 mg/kg). Escalating doses of adenosine resulted in progressive heart rate slowing until maximum slowing occurred. The adenosine doses required to reach maximum slowing of heart rate were 118 +/- 24, 410 +/- 197, 620 +/- 256, 855 +/- 264, 944 +/- 329 and 1062 +/- 463 (SD) micrograms at theophylline doses of 0, 10, 20, 30, 40, and 50 mg/kg, respectively, which were correlated (r2 = .57, p < .001). At baseline, increasing theophylline doses produced a progressive reduction in left ventricular systolic pressure. However, adenosine at maximum slowing doses reversed this effect where left ventricular systolic pressure at 0 mg/kg of theophylline was similar to left ventricular systolic pressure at 50 mg/kg of theophylline (p < .05). At baseline, 10 to 50 mg/kg of theophylline had no significant effect on +dP/dtmax. However, adenosine (at maximum slowing) decreased +dP/dtmax before theophylline administration (p < .01). Theophylline at doses of 10 to 50 mg/kg reversed adenosine's negative inotropic effect, where +dP/dtmax values at maximum slowing were greater than values at 0 mg/kg of theophylline (p < .05). CONCLUSIONS: Adenosine can reverse theophylline-induced electrophysiologic and hemodynamic instability, whereas theophylline can reverse the negative inotropic actions of adenosine. However, higher adenosine doses are needed to elicit these changes as the theophylline dose increases.

Reduced renal accumulation and toxicity of cisplatin in experimental galactosemia.

The kidneys of streptozotocin (STZ)-diabetic rats are resistant to certain toxic effects of the antineoplastic drug cisplatin. The mechanism is unknown. This study used the galactosemic rat model to test the hypothesis that the apparent diabetes-induced protection is due to changes in the kidney secondary to chronically elevated hexose concentrations. Galactosemic rats are normoinsulinemic and are free from many of the multiple biochemical abnormalities seen in STZ diabetics. The experiments compared renal cortical platinum (Pt) and blood urea nitrogen (BUN) levels after intraperitoneal injection of 5 mg/kg of cisplatin in galactosemic, STZ-diabetic, and age-matched nondiabetic Sprague-Dawley rats. Nephrotoxicity was defined as a BUN concentration ratio (after to before cisplatin) > 2.5. The results demonstrate that the kidneys of both galactosemic and STZ-diabetic rats became resistant to cisplatin-induced elevation of BUN and, further, that the development of the protection was related to the duration of the diabetic state. Although the protective effect developed more slowly in the galactosemic rats, the attenuation of the rise in BUN was ultimately comparable to that seen in STZ diabetics. Renal cortex [Pt] after cisplatin injection was significantly lower in galactosemics and STZ diabetics compared with age-matched nondiabetics, with the order nondiabetics > galactosemics > STZ diabetics. It was noted, however, that renal Pt accumulation was maximally depressed within 4 weeks of experimental diabetes, whereas the BUN ratio continued to decline with increasing duration of both galactosemia and STZ diabetes. Thus, reduced renal Pt accumulation cannot by itself explain the progressive attenuation of the toxicity. The results support the hypothesis and suggest that the galactosemic rat will be a useful model for mechanistic study of diabetes-induced protection from cisplatin nephrotoxicity.

Brain norepinephrine reductions in soman-intoxicated rats: association with convulsions and AChE inhibition, time course, and relation to other monoamines.

The organophosphate chemical nerve agent, soman, causes convulsions, neuropathology, and, ultimately, death. A major problem in treating soman intoxication is that peripherally acting pharmacological agents which prevent death do not prevent seizures. Although a primary cause of these symptoms is the excess of acetylcholine which follows acetylcholinesterase (AChE) inhibition, centrally acting muscarinic blockers, such as atropine, alleviate, but do not block, the convulsive actions of soman. Moreover, there is a relatively weak relationship between CNS reductions of AChE and the incidence of convulsions. There is evidence suggesting that soman intoxication stimulates the release of norepinephrine (NE) in the brain. Recent evidence has implicated NE in the induction and/or maintenance of seizures. Thus, in the present study the relations among soman-induced convulsions, AChE inhibition, and brain NE and other monoamine changes were examined. The time course of brain NE recovery was also determined. Rats were injected (im) with a single dose (78 micrograms/kg) of soman. At this dose 68% of the injected rats developed convulsions. Both convulsive and nonconvulsive rats were sacrificed between 1 and 96 h following soman injection and NE levels in the rostral forebrain and olfactory bulb were determined by HPLC with electrochemical detection. In all convulsive rats NE levels declined substantially. Forebrain NE levels were decreased by 50% at 1 h and 70% at 2 h following soman injection. Recovery of NE began at 8 h and was complete by 96 h following soman administration. Although nonconvulsive rats showed other signs of intoxication, NE levels in these rats were unchanged. Dopamine (DA) and serotonin (5-HT) levels were not significantly affected in either convulsive or nonconvulsive rats. However, 5-hydroxyindoleacetic acid, the major metabolite of 5-HT, and homovanillic acid and 3,4-dihydroxyphenylacetic acid, the two major metabolites of DA, were increased significantly in the forebrain of convulsive, but not nonconvulsive rats, indicating an increase in 5-HT and DA turnover. However, in contrast to the abrupt decline in NE, these increases in DA and 5-HT metabolites were slow and progressive. Taken together, the present results and other recent findings suggest that rapid, sustained NE release could play a role in the induction and/or maintenance of soman-induced convulsions, whereas increased release of 5-HT and DA may be a consequence of seizures. Further investigation of the role of NE in soman-induced convulsions may lead to improved treatment of soman intoxication and a better understanding of the role of NE in other forms of seizures, including human epilepsy.

Exploring the hexokinase glucose binding site through correlation analysis and molecular modeling of glucosamine inhibitors.

A series of N-substituted glucosamines has been designed, synthesized, and tested as inhibitors of yeast hexokinase. All derivatives exhibited competitive inhibition kinetics with respect to glucose. Quantitative structure-activity relationships were derived from the resulting inhibition data. The most significant equation demonstrated the existence of highly specific steric effects for the seven meta-substituted benzoylglucosamines included in the relationship. Molecular modeling of potential complexes between the inhibitors and the hexokinase substrate binding site strongly suggests that the steric effects arise from potential contacts with two amino acid residues lying in the region occupied by the amide substituents.

Progressive and concurrent deterioration of vagus-stimulated and hypoglycemia-induced glucagon secretion in streptozotocin-diabetic rats.

The effects of left cervical vagus nerve stimulation on glucagon secretion were studied in streptozotocin-diabetic and age-matched control adult male rats. At two-week intervals, after the induction of streptozotocin-diabetes, streptozotocin-diabetic and age-matched control rats were anesthetized with chloral hydrate (350 mg/kg, ip). Left cervical vagus nerves were electrically stimulated via a Grass stimulator with 5-volt monophasic pulses of 3 msec duration at a frequency of 20 Hz for 1, 2, and 4 min. Arginine-induced glucagon secretion was also determined. Vagus nerve-stimulated (2 and 4 min) glucagon secretion deteriorated as the duration of streptozotocin-diabetes increased. Glucagon secretion in response to vagus nerve stimulation was virtually absent by 12 weeks of streptozotocin-diabetes. However, arginine-induced glucagon secretion was unaffected. Subsequent experiments showed that the defect in glucagon secretion from vagal stimulation occurred concurrently with that seen from insulin-induced hypoglycemia. These results indicate that the impaired hypoglycemia-induced glucagon secretion in long-term streptozotocin-diabetic rats may be correlated with the deterioration of the parasympathetic nervous system transmission in streptozotocin-diabetes.

On the specificity of the acetylcholinesterase defect in dystrophic mice.

The tetrameric form of acetylcholinesterase (AChE) in ReJ/129 dystrophic mice was demonstrated to be absent from endplate-poor regions of skeletal muscle but present in endplate-rich regions. Skeletal muscle secreted normal amounts of this form of AChE. Visceral organs had normal amounts and distribution of the AChE molecular forms. These results suggest that the AChE defect in dystrophic mice is limited to skeletal muscle, and the defect does not reflect an abnormality of AChE synthesis but probably reflects an inability to incorporate the enzyme into skeletal muscle membranes.

Distribution and solubilization of the molecular forms of acetylcholinesterase in rat urinary bladder and sphincter.

The distribution and solubility of the molecular forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) were examined in adult, male rat bladder body and sphincter. Four distinct forms of the enzyme solubilized from bladder body were separated on sucrose density gradients. Two of the forms (A8 and A12) corresponded to asymmetric proteins and were solubilized with high ionic strength buffer. The other two forms represented globular forms. The smallest form (G1) was soluble in low ionic strength buffer without detergent. About 33% of the larger globular form (G4) required detergent for solubilization. There were only minor differences in the distribution of these forms in juvenile rat bladder and adult, female rat bladders. Sphincter tissue lacked one of the asymmetric forms but otherwise resembled the bladder bodies. These results demonstrate that some smooth muscle organs have significant amounts of asymmetric, as well as globular, forms of acetylcholinesterase and suggest that additional studies should be performed to characterize the enzyme in this and other smooth muscle systems.

Effects of chronic sodium salicylate feeding on the impaired glucagon and epinephrine responses to insulin-induced hypoglycaemia in streptozotocin diabetic rats.

The potential role of endogenous prostaglandins in glucagon and epinephrine responses to insulin-induced hypoglycaemia was studied in streptozotocin-diabetic and age-matched control adult male rats. Rats made diabetic with a single intravenous injection of streptozotocin (65 mg/kg) developed impaired glucagon and epinephrine responses to insulin-induced hypoglycaemia by 80-100 days. Plasma glucagon levels in response to insulin-induced hypoglycaemia in streptozotocin-diabetic rats (167 +/- 67 pg/ml) were significantly lower (p less than 0.01) than those in control rats (929 +/- 272 pg/ml). Similarly, plasma epinephrine levels in hypoglycaemic state in streptozotocin-diabetic rats (11 +/- 8 pmol/ml) were also significantly lower (p less than 0.01) compared to control rats (37 +/- 13 pmol/ml). Streptozotocin-diabetic rats provided with sodium salicylate (25 mg/100 ml) in their drinking water from day one of diabetes exhibited prevention of the blunted glucagon and epinephrine responses to insulin-induced hypoglycaemia. About 80-100 days after the chronic sodium salicylate treatment in streptozotocin-diabetic rats, both plasma glucagon levels (1080 +/- 169 pg/ml) and plasma epinephrine levels (39 +/- 8 pmol/ml) were essentially identical to plasma glucagon levels (1074 +/- 134 pg/ml) and plasma epinephrine levels (37 +/- 5 pmol/ml) in control rats in hypoglycaemic state. These animals also exhibited an improvement in the diabetic state in that they had less severe hyperglycaemia and lack of weight gain. These results suggest that the blunted glucagon and epinephrine responses to insulin-induced hypoglycaemia may be related to altered prostaglandin levels in streptozotocin-diabetes.

Maintenance by glycyl-L-glutamine in vivo of molecular forms of acetylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.

The 24-hr intracarotid infusion of plasma-treated glycyl-L-glutamine (3 microM) produced significant enhancement of the monomeric G1 and tetrameric G4 forms of acetylcholinesterase of the cat superior cervical ganglion 48 hr after denervation, in comparison with denervated, noninfused controls. No significant effect of glycyl-L-glutamine could be demonstrated 4 or 6 days after denervation. These findings are consistent with the conclusion, drawn from a previous in vitro study, that glycyl-L-glutamine acts at a stage prior to the aggregation of the G1 form into higher polymers to maintain the acetylcholinesterase content of denervated ganglia. It is proposed that the dipeptide may regulate the transcription of the DNA for acetylcholinesterase to its corresponding mRNA.

Diabetes and diuretic-induced alterations in function of rat urinary bladder.

Studies were done to characterize the bladder dysfunction associated with diabetes mellitus and to distinguish between changes occurring from increased diuresis and autonomic neuropathy. Four experimental conditions were compared: control, 4-wk-streptozocin-induced diabetes, sucrose feeding (diuretic), and galactose feeding (diuretic and sugar alcohol). A 10-fold increase in urine output and 25-50% increases in bladder weight, protein content, and DNA content were observed in all noncontrol treatment groups. Compliance properties were studied by measuring the intravesicular pressure as the bladder was infused with buffer in vitro. All treated bladders exhibited a reduction in plateau pressure and an increase in fluid capacity. Thus, diuresis results in an increased bladder size, which correlates with an alteration of compliance properties. Nervous system control in anesthetized rats was examined by monitoring contractions as the bladder was infused with buffer. Three distinct patterns of response were observed: normal, diabetic, and diuretic (galactose and sucrose treatments). The difference between responses in diuretic and diabetic animals suggests the presence of a diabetes-induced alteration in nerve regulation of the bladder. Reserpine pretreatment of control or diuretic models produced marked changes in the pattern of contractions, whereas pretreatment of diabetic rats had only modest effects. This suggests that diabetic bladders were lacking sympathetic control before the drug treatment. When rats treated for 4 wk with galactose were removed from this diet for 4 wk before testing, the bladders responded similarly to controls. This observation, coupled with the fact that galactose did not produce the same response as diabetes in the in vivo experiment, suggests that the galactose model does not produce irreversible functional neuropathies.

Regional noradrenergic and cholinergic neurochemistry in the rat urinary bladder: effects of age.

Neurochemistry of the base and body of the rat urinary bladder was compared for both adrenergic and cholinergic parameters using Fischer 344 rats. In bladder base and body, respectively, the concentration (pmol./mg. wet weight) of norepinephrine was 23.4 and 2.16, of acetylcholine was 26.7 and 18.3, and of choline was 96.7 and 199. The activity (nmol./mg. protein/hour) of tyrosine hydroxylase was 422 and less than 50, of monoamine oxidase was 80.6 and 126, of choline acetyltransferase was 17.4 and 11.5, and of acetylcholinesterase (nmol./mg. wet weight/hour) was 485 and 165. Treatment with alpha-methyl-p-tyrosine did not alter norepinephrine concentration in bladder base but decreased it by 27% in bladder body. Studies were also done to determine whether age-related changes exist in the adrenergic and cholinergic neurochemistry of the rat urinary bladder. Bladders from rats of 6-7, 15-17, and 22-24 mo. of age were examined. The only age-related differences noted were a progressive decrease in level of monoamine oxidase activity in both bladder regions and an increase in bladder base norepinephrine concentration from 6-7 to 15-17 mo. followed by a decrease at 22-24 mo. Overall, the results show marked regional variations in bladder neurochemistry which remain remarkably stable as the animals grow old.

The molecular forms of acetylcholinesterase in rat thymus.

Using sucrose density gradients, studies on the molecular forms of AChE in rat thymus show the presence of the 4S and 10S enzyme but the absence of the 16S form--which is itself a characteristic form in rat skeletal muscle. Additionally, there are variations in solubility characteristics and in developmental aspects between rat skeletal muscle and rat thymus.

Acetylcholinesterase molecular forms in serum and erythrocytes of laboratory animals.

The distribution of the molecular forms of acetylcholinesterase in the blood of various animal species was examined. The globular tetrameric form was most frequently observed in serum but mouse serum also contained a globular monomer. Globular monomers (rat) dimers (mouse, dog, rabbit) and tetramers (dog) were found in erythrocytes. Interspecies differences make it difficult to formulate a cohesive theory as to the origin and function of blood-borne enzymes.

The acetylcholinesterase abnormality in dystrophic mice is a reflection of a maturational defect.

Juvenile mice of the ReJ/129 strain exhibit a distribution of acetylcholinesterase molecular forms that is similar to the pattern previously observed in dystrophic mice. The change to an adult distribution of the enzyme occurs at about 3 weeks of age which is also when dystrophic signs first become evident. It is suggested that dystrophic mouse muscle fails to mature with respect to the molecular forms of acetylcholinesterase.