Racial differences in platelet cytosolic calcium and calcitropic hormones in normotensive subjects.

Elevated levels of serum parathyroid hormone (PTH) and platelet cytosolic free calcium ([Ca2+]i) have been reported in subjects with essential hypertension. In addition, there is a positive correlation between serum PTH and platelet [Ca2+]i in white subjects with essential hypertension. Black normotensive subjects have relatively higher levels of serum PTH when compared to white normotensive subjects. To investigate the possibility that elevated serum PTH levels in black normotensives may contribute to elevated platelet [Ca2+]i, calcitropic hormone profiles and platelet [Ca2+]i were determined in 31 black normotensive subjects and 34 age-matched white normotensive subjects. There was no difference between the two groups in total serum calcium, plasma ionized calcium, or creatinine clearance. However, serum PTH was significantly elevated (P < .02) in the black normotensive group. Serum 1,25(OH)2 vitamin D levels were similar between the two groups whereas serum 25(OH) vitamin D levels were significantly lower (P < .001) in the blacks. The 24 h urinary excretion of Ca was also lower (P < .05) in the black normotensive group. Basal platelet [Ca2+]i was significantly lower (P < .05) in black normotensive than in white normotensive subjects. Serum PTH levels did not correlate with platelet [Ca2+]i in either group, or in the groups combined. These results demonstrate that the higher serum PTH concentrations in black normotensives is not associated with higher platelet [Ca2+]i, as is the case in white hypertensive patients.

Parathyroid hormone, platelet calcium, and blood pressure in normotensive subjects.

Relations between platelet cytosolic calcium, parathyroid hormone, and blood pressure were investigated in 91 normotensive subjects: 47 men and 44 women ranging in age from 24 to 70 years. The men had higher mean arterial blood pressure, serum creatinine, and body mass index than the women. Serum total calcium, plasma ionized calcium, and parathyroid hormone (measured as both intact hormone and mid-molecule fragment) were not different between men and women; however, serum phosphate was higher in women than in men. Basal platelet cytosolic calcium was higher in men than in women (113.7 +/- 1.9 versus 105.9 +/- 1.7, respectively; p less than 0.01), but there was no difference in the peak platelet cytosolic calcium responses to thrombin between the two groups. In the combined group of male and female subjects, platelet cytosolic calcium correlated with diastolic blood pressure and mean arterial pressure (r = 0.37, p less than 0.001 and r = 0.32, p less than 0.01, respectively). Intact parathyroid hormone correlated with systolic and mean arterial blood pressure (r = 0.41, p less than 0.001 for both). Age correlated with both systolic blood pressure (r = 0.40, p less than 0.001) and intact parathyroid hormone (r = 0.51, p less than 0.001). When multiple regression analysis was performed using mean arterial pressure as the dependent variable, platelet cytosolic calcium and intact parathyroid hormone maintained significant correlations with mean arterial pressure. Platelet cytosolic calcium did not correlate with intact parathyroid hormone. These results suggest that both platelet cytosolic calcium and intact parathyroid hormone are associated with blood pressure regulation in normotensive subjects. However, the influences of these two factors on blood pressure are not interrelated.

Temperature alters ethanol-induced fluidization of C57 mouse brain membranes.

The interaction between temperature and ethanol-induced fluidization was investigated in brain synaptic plasma membranes from C57BL/6 mice. Changes in fluidity were measured using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Fluorescence polarization was tested in the presence and absence of ethanol at 25, 32 and 37 degrees C. An increase in temperature resulted in a significant increase in the baseline fluidity of the membranes and an increase in the magnitude of ethanol-induced fluidization of brain membranes. The combined effect of temperature on baseline fluidity and the magnitude of the response to ethanol resulted in a significant temperature-related increase in the relative response to ethanol (% change in polarization). The minimum concentration of ethanol required to cause a significant increase in the fluidity of the membranes was 170.7 mM at 25 degrees C and 85.3 mM at both 32 and 37 degrees C. The present results indicate that temperature-related changes in the effects of ethanol on membrane properties may underlie the effects of temperature on ethanol sensitivity in C57 mice.

Calcitropic hormones, platelet calcium, and blood pressure in essential hypertension.

Plasma ionized calcium, platelet cytosolic calcium (using the fura-2 method in gel-filtered platelets), parathyroid hormone (both the intact hormone and a midmolecule portion), calcitriol, and calcidiol were measured in 19 untreated male patients with essential hypertension and 19 age-matched normotensive male research subjects. Mean levels of platelet cytosolic calcium, parathyroid hormone, calcitriol, and calcidiol were all significantly higher, whereas plasma ionized calcium was significantly lower, in the hypertensive group compared with the normotensive group. Both platelet cytosolic calcium and intact parathyroid hormone were positively correlated with mean arterial pressure (r = 0.58, p less than 0.001; r = 0.54, p less than 0.001, respectively), whereas plasma ionized calcium was inversely correlated with mean arterial pressure (r = -0.60, p less than 0.001) in the combined group of all study subjects. All three of these correlations were significant in the hypertensive group alone but not in the normotensive group alone. When analyzed with plasma ionized calcium, body mass index, serum calcitriol, and calcidiol in a multivariable regression model, the significance of the partial regressions of platelet cytosolic calcium and parathyroid hormone with mean arterial pressure persisted. Intact parathyroid hormone was positively correlated to platelet cytosolic calcium (r = 0.43, p less than 0.01) and plasma ionized calcium was inversely correlated to platelet cytosolic calcium (r = -0.44, p less than 0.01). These results confirm previous reports of disturbances of calcium metabolism in essential hypertension and suggest that the elevated platelet cytosolic calcium observed in essential hypertension may be linked to one or more of these alterations of calcium metabolism.

The effect of chronic ethanol on glutamate binding in human and rat brain.

Quantitative autoradiographic techniques demonstrate that chronic alcohol administration causes a decrease in [3H]-glutamate binding to hippocampal N-methyl-D-aspartate (NMDA) receptors. A 14% decrease in [3H]-glutamate binding in the hippocampal CA1 region is seen both in the rat after five days of ethanol administration and in postmortem hippocampal tissues from alcoholics. In the rat, 24 hr ethanol withdrawal values are intermediate between control and alcohol binding levels. There was no significant effect of ethanol on [3H]-glutamate binding in the cortex or caudate.

Studies on the interaction between ethanol and amfonelic acid.

Amfonelic acid (AFA), a non-amphetamine central stimulant dose-dependently reduced the hypnotic effect of ethanol in C57B1/6 mice. It did not enhance the elimination of ethanol. Amfonelic acid failed to modify the duration of pentobarbitone-induced hypnosis or the ethanol-induced hypothermia in these animals. Combined treatment with amfonelic acid and a lipophilic alpha 1-adrenoceptor agonist was not more effective than amfonelic acid alone in blocking ethanol hypnosis. The stimulation of locomotor activity by amfonelic acid in C57B1/6 mice was more sensitive to the blocking effect of ethanol than stimulation induced by d-amphetamine. The blocking effect of amfonelic acid, but not that of d-amphetamine, on the effects of ethanol developed tolerance. In pimozide-pretreated mice, amfonelic acid failed to reduce the ethanol-induced hypnosis. Hence it appears that dopamine (DA) released by amfonelic acid is responsible for its antagonism of ethanol. However, though amfonelic acid acted as a strong releaser of DA in Swiss-Webster, CD-1, DBA-2 and BALB/c mice, in these strains it failed to reduce the effect of ethanol. Moreover, methylphenidate, a dopaminergic stimulant, which acts by a mechanism similar to that of amfonelic acid was not effective in reducing the hypnotic effect of ethanol in C57B1/6 mice. For these reasons, additional mechanisms may have to be considered to explain this strain-dependent effect of amfonelic acid.

Modulation of serotonin receptors by specific phosphatidylcholines.

Treatment of mouse cortical brain membranes with dioleoylphosphatidylcholine produced a large (50%) decrease in serotonin binding sites. The time course for this effect paralleled an increase in oleic acid in membrane phosphatidycholine and an increase in membrane fluidity. "Active Lipid" produced a similar decrease in serotonin binding sites, while fluidizing the membranes even more strongly. Distearoylphosphatidylcholine had no effect on serotonin binding sites or membrane fluidity by itself, but was capable of counteracting both the reduction in binding sites and membrane fluidity produced by "Active Lipid". The data indicate that specific phosphatidylcholines can have profound effects on serotonin receptors, but a clear picture of the relative importance of membrane fluidity per se versus more specific phospholipid effects will require further investigation.

Rapid visualization of proteins in isoelectric focusing gels: isolation of brain tubulin subspecies.

A method for visualization of unmodified proteins in polyacrylamide isoelectric focusing (IEF) gels is described. The proteins appear as white, translucent bands when disc IEF gels are placed in water. The gel can be scanned with a scanning spectrophotometer, or the protein bands can be excised using a simple apparatus, which is described, and the protein eluted. The method is fast, selective for proteins, sensitive, and quantitative. It has been used to isolate tubulin species separated by as little as 0.5 mm in disc IEF gels.

Regional effects of ethanol on glutamate levels, uptake and release in slice and synaptosome preparations from rat brain.

Chronic ethanol treatment effects a decrease in K+-stimulated endogenous glutamate release and an increase in glutamate levels in rat frontal cortex. This treatment also increases (3H)-glutamate uptake in striatal and hippocampal slices. No uptake changes were observed in either cortical slices or synaptosomes. Ethanol withdrawal increases K+-stimulated glutamate release in cortex and hippocampus and glutamate levels in cortex, striatum and hippocampus. (3H)-glutamate uptake is increased in striatum of withdrawn animals. Thus, regional variations were observed with regard to both effect and degree of effect. Because in vitro and acute ethanol treatments had no effect on uptake or release, these changes probably result from activated feedback mechanisms that attempt to compensate for ethanol's action on brain systems rather than direct effects on membrane structure. Combined level-release data suggest an effect of ethanol on glutamate distribution in cortex and striatum. Combined uptake and release data indicate a correlation between diminished glutamate synaptic activity and the addicted state, and between heightened glutamate synaptic activity and the withdrawn state.

Brain tubulin microheterogeneity in the mouse during development and aging.

Mouse brain tubulin was analyzed on isoelectric focusing gels. High-resolution gels utilizing Bio-Rad ampholytes (pH 4--6) revealed 5--6 bands in the region corresponding to the alpha-subunit of tubulin and 10 or more for the beta-subunit. The same general banding pattern was observed regardless of the method of preparation of the tubulin. Two species prominent in the brains of immature mice, alpha 6 and beta 2, virtually disappeared during maturation, while species beta 6 to beta 10 appeared. No significant changes from the mature pattern were seen during aging (examined at 12, 23, and 30 months of age).

Serotonin-sensitive adenylate cyclase activity of immature rat brain.

Cell-free preparations from superior and inferior colliculi of very young rats (1-3 days old) contained adenylate cyclase systems which were highly responsive to serotonin. The response to serotonin declined markedly during early development and was very low at maturity. Adenylate cyclase activity in the 10,000 times g particulate fraction from colliculi of newborn rats was significantly stimulated by 0.05 muM serotonin. Half-maximal activation was produced with less than 1 muM serotonin. Maximal stimulation of collicular adenylate cyclase was about 80% above basal enzyme activity and occurred with approximately 50 muM serotonin. Tryptamine and several derivatives of serotonin produced responses which were comparable to that obtained with serotonin; 5-methoxytryptamine was uniformly the most active compound tested. Norepinephrine or dopamine produced much smaller increases in adenylate cyclase activity. Stimulation of collicular adenylate cyclase by serotonin was significantly but incompletely blocked by serotonin antagonists, including d-lysergic acid diethylamide (d-LSD), 2-bromo-d-lysergic acid diethylamide, methysergide, 1-methyl-8 beta-carbobenzyloxy-aminomethyl-10 alpha-ergoline and cyproheptadine. Chlorpromazine also produced partial blockade. In contrast, l-lysergic acid diethylamide, haloperidol, propranolol, phenoxybenzamine and morphine were ineffective as serotonin blocking agents. Of the compounds which produced a partial blockage of serotonin action, d-LSD, cyproheptadine and chlorpromazine were themselves capable of stimulating adenylate cyclase activity. These results are consisent with the existence of multiple receptors in rat brain which are capable of interacting with indoleamines.