Cerebrospinal fluid levels of amyloid beta-protein in Alzheimer's disease: inverse correlation with severity of dementia and effect of apolipoprotein E genotype.

Alzheimer's disease (AD) is characterized by formation in brain of neurofibrillary tangles and of amyloid deposits. The major protein component of the former is tau, while the latter are composed of amyloid beta-peptides (A beta), which are derived by proteolytic cleavage of the amyloid beta-protein precursor (APP). Both tau and various secretory APP derivatives including A beta and APPS are present in human cerebrospinal fluid (CSF). To investigate whether clinical signs of AD are paralleled by changes in CSF levels of these proteins, we correlated quantitative measures of dementia severity with CSF concentrations of A beta, of APPS, and of tau. We found that levels of A beta in CSF of AD patients were inversely correlated both to cognitive and to functional measures of dementia severity. In contrast, levels of APPS and of tau did not correlate with dementia severity. Apolipoprotein E (apoE) genotype did not influence CSF levels of A beta, APPS, or tau, which were similar among AD patients with Apo E epsilon 3/3, epsilon 3/4, and epsilon 4/4 alleles. These data indicate that CSF levels of A beta decrease with advancing severity of dementia in AD and suggest that they are independent of a patient's Apo E genotype.

Dioctanoylglycerol stimulates accumulation of [methyl-14C]choline and its incorporation into acetylcholine and phosphatidylcholine in a human cholinergic neuroblastoma cell line.

Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [14C]choline uptake in cultured human neuroblastoma (LA-N-2) cells. As this effect has not, to our knowledge, been reported before, it was of interest to characterize it in more detail. In the presence of 500 microM dioctanoylglycerol the levels of [14C]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [14C]acetylcholine and long fatty acyl chain [14C]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [14C]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent Km for choline uptake to 56 +/- 9% of control (n = 4). The Vmax was not significantly altered. The stimulation of [14C]choline accumulation by dioctanoylglycerol was not dependent on protein kinase C activation; the effect was not mimicked by phorbol ester or by 1-oleoyl-2-acetylglycerol, and was not inhibited by the protein kinase C inhibitors H-7 or staurosporine, or by prolonged pretreatment with phorbol 12-myristate 13-acetate. The effect of dioctanoylglycerol was slightly (but not significantly) reduced by EGTA and strongly inhibited by the cell-permeant calcium chelator bis(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)ester. Although these results implicate elevated intracellular calcium in the response, dioctanoylglycerol did not increase phosphatidylinositol hydrolysis in LA-N-2 cells, and its effect was not inhibited by the diacylglycerol kinase inhibitor R 59 022 (which blocks the conversion of diacylglycerol to phosphatidic acid, a known stimulator of phosphatidylinositol hydrolysis).(ABSTRACT TRUNCATED AT 250 WORDS)

Base-exchange and cell growth in 3T6 mouse fibroblasts.

Synthesis of phosphatidyl serine by base-exchange and DNA synthesis both declined rapidly in mouse fibroblast cultures upon withdrawal of serum from nutrient medium. Base-exchange returned to control levels within 3 h after the addition of serum, while DNA synthesis lagged by 10 h. Phosphatidyl serine synthesis is thus an early response of 3T6 cells to mitogenic signals.

Relationship between pituitary ACTH content and hypothalamic catecholamines in the rat.

Hypothalamic concentrations of epinephrine and norepinephrine were determined in rats following 6-hydroxydopamine lesions of the locus coeruleus and subcoeruleus system and following sham-operation. These concentrations were correlated with pituitary ACTH content. While the lesion procedure did not have a major effect on hypothalamic monoamine levels, we were able to demonstrate a strong negative correlation between hypothalamic epinephrine and pituitary ACTH content independent of the experimental condition. Only a weak negative correlation was observed for hypothalamic norepinephrine and pituitary ACTH. Our recent and previous data suggest a tonic and phasic inhibition of ACTH release by hypothalamic monoamines.

Acetylcholine synthesis and secretion by LA-N-2 human neuroblastoma cells.

We have investigated the rates of acetylcholine (ACh) synthesis and release in LA-N-2 cells in order to characterize them as a potential model of cholinergic neurons. When grown in a serum-containing medium the cells extend few neurites. In the absence of serum most cells develop processes. ACh content of the cells (determined by a radioenzymatic assay) varies with extracellular choline concentration in a saturable fashion, reaching a maximum of approximately 25 nmol/mg protein. Radiolabeled choline is taken up by the cell and converted to ACh or phosphocholine, as determined by purification from cell extracts by HPLC, in a saturable manner which is described by a single rectangular hyperbola. Hemicholinium-3 (100 microM) inhibits this uptake. The cells release ACh spontaneously and this release is enhanced upon depolarization with potassium or veratridine (the latter effect is blocked by tetrodotoxin). The data demonstrate that LA-N-2 cells exhibit some properties similar to cholinergic neurons and may therefore be useful for studies of ACh synthesis and release.

Synthesis of acetylcholine from choline derived from phosphatidylcholine in a human neuronal cell line.

Cholinergic neurons are unique among cells since they alone utilize choline not only as a component of major membrane phospholipids, such as phosphatidylcholine (Ptd-Cho), but also as a precursor of their neurotransmitter acetylcholine (AcCho). It has been hypothesized that choline-phospholipids might serve as a storage pool of choline for AcCho synthesis. The selective vulnerability of cholinergic neurons in certain neurodegenerative diseases (e.g., Alzheimer disease, motor neuron disorders) might result from the abnormally accelerated liberation of choline (to be used as precursor of AcCho) from membrane phospholipids, resulting in altered membrane composition and function and compromised neuronal viability. However, the proposed metabolic link between membrane turnover and AcCho synthesis has been difficult to demonstrate because of the heterogeneity of the preparations used. Here we used a population of purely cholinergic cells (human neuroblastoma, LA-N-2), incubated in the presence of [methyl-3H]methionine to selectively label PtdCho synthesized by methylation of phosphatidylethanolamine, the only pathway of de novo choline synthesis. PtdCho, purified by thin-layer chromatography, contained 90% of the label incorporated into lipids, demonstrating that LA-N-2 cells contained phosphatidylethanolamine N-methyltransferase. Three peaks of radioactive material that cochromatographed with authentic Ac-Cho, choline, and phosphocholine were observed when the water-soluble metabolites of the [3H]PtdCho were purified by high-performance liquid chromatography. Their identities were ascertained by subjecting them to enzymatic modifications with acetylcholinesterase, choline oxidase, and alkaline phosphatase, respectively. The results demonstrate that AcCho can be synthesized from choline derived from the degradation of endogenous PtdCho formed de novo by methylation of phosphatidylethanolamine.

Phosphatidylcholine as a precursor of choline for acetylcholine synthesis.

It has been hypothesized that the selective vulnerability of certain brain cholinergic neurons in Alzheimer's disease may reflect the unique way that choline is utilized by these neurons, i.e. not only as a component of major membrane phospholipids, e.g. phosphatidylcholine (PC), but also as a precursor of their neurotransmitter, acetylcholine (ACh). A prolonged utilization of choline liberated from PC, for ACh production, without adequate resynthesis of this lipid, might result in a net loss of the phosphatide followed by an impairment of membrane function and loss of cellular viability. Studies described in this paper, performed on electrically stimulated striatal slices and on cholinergic cell lines, test this hypothesis. 1) Electrically-stimulated striatal slices continue to release ACh, and sustain their free choline and ACh levels, even when perfused with a choline-free medium. Striatal levels of PC decline under these circumstances, and this decline can be blocked by adding tetrodotoxin (which blocks neuronal depolarization) or choline to the medium. The other major membrane phospholipids, phosphatidylserine and phosphatidylethanolamine, also decline proportionately to PC when slices are stimulated in the absence of choline. 2) In a population of purely cholinergic cells (human neuroblastoma, LA-N-2), ACh can be synthesized from choline derived from degradation of endogenous PC formed de novo by methylation of phosphatidylethanolamine. 3) PC content of cells in culture (neuroblastoma X glioma hybrid, NG 108-15) can be altered by adding various amounts of choline to the growth media. The proportion of PC in the cells apparently affects cellular survival and rate of growth. Taken together these data demonstrate that cholinergic neurons utilize the choline stored in PC to synthesize ACh; that this process may lead to a depletion in membrane phospholipids (when choline supply is inadequate); and that the resulting changes in neuronal membrane composition might adversely affect cellular viability.

Multiple classes of calcium channels in mouse pituitary tumor cells.

Synthetic corticotropin-releasing factor (CRF) is a potent adrenocorticotropin (ACTH) secretagogue in the mouse pituitary tumor cell strain AtT20/D16v (D16). In the absence of added calcium in the incubation medium a dose of 5 nM CRF stimulates ACTH secretion 2-fold over control values while at medium calcium concentrations greater than 1 mM the same dose of CRF elicits a 3-fold stimulation. In the presence of EGTA or of the calcium antagonists verapamil, cobalt, or lanthanum the CRF effect is abolished. Depolarizing concentrations of extracellular K+ lead to a rapid increase in cell-associated calcium, a response which is inhibited by the dihydropyridine calcium antagonist nimodipine. Although treatment with CRF does not alter the concentration of cell-associated calcium in D16 cells, ACTH secretion stimulated by both CRF and elevated medium K+ are inhibited by nimodipine in a dose-related manner. The results indicate that D16 cells possess both voltage-sensitive and CRF-activated calcium channels.

ACTH secretion in mouse pituitary tumor cells in culture: inhibition of CRF-stimulated hormone release by somatostatin.

Synthetic corticotropin-releasing factor (CRF) stimulates ACTH secretion in the clonal mouse pituitary cell strain AtT20/D16v (D16) in a dose-dependent manner with a half-maximal effect at 2X10(-9)M. A single dose of 5X10(-9)M CRF maximally stimulates the rate of ACTH secretion during the initial two hrs of treatment. During the period of maximal CRF stimulation intracellular hormone concentration declines progressively to a nadir at 4 hrs. During the ensuing 24 hrs of incubation intracellular hormone levels in CRF-stimulated cells increase gradually toward control values. Somatostatin (SRIF) inhibits the secretory response to CRF. This action of SRIF is dose-dependent with a half-maximal effect at 1X10(-9)M and results in decreased maximal ACTH secretion with little effect on the ED50 for CRF.

Adrenocorticotropin secretion by mouse pituitary tumor cells in culture: the role of Ca+2 in stimulated and somatostatin-inhibited secretion.

SRIF inhibits ACTH secretion by AtT20/D16v (D16) mouse pituitary cells stimulated by high (50 mM) extracellular concentrations of K+ or by divalent cation ionophores. Although stimulation of ACTH secretion by K+ requires extracellular Ca+2, the response is invariant over medium Ca+2 concentrations of 0.003-1 mM; with Ca+2 concentrations from 1-5 mM there is a dramatic amplification of the secretory response. SRIF at concentrations of 10(-8) M completely inhibits the secretory response to K+ at Ca+2 concentrations between 0.2 and 1 mM; with increasing medium Ca+2 above 1 mM there is a progressive attenuation of SRIF-inhibition. At concentrations of 5 mM, Ca+2 alone can serve as an ACTH secretagogue. The ionophore ionomycin stimulates ACTH secretion in a Ca+2-dependent manner with a half-maximal effect at 5 X 10(-6) M ionomycin. The secretory response to ionomycin and to X537A is inhibited by at least 50% by SRIF. The secretory response to K+ is accompanied by a rapid and sustained increase in 45Ca+2 uptake, whereas the ionophores ionomycin, X537A, and A23187 increase Ca+2 efflux. SRIF does not affect Ca+2 movement across D16 cell membranes in response to either K+ or ionophores. These results show that an increase in intracellular Ca+2 is an effective stimulus to ACTH secretion by D16 cells and inhibition of ACTH secretion by SRIF is not effected by interference with the stimulus-elicited increase in intracellular Ca+2.

Inhibition of adrenocorticotropin secretion by somatostatin in pituitary cells in culture.

AtT20/D16v is a clonal strain of mouse pituitary tumor cells which synthesizes and secretes ACTH. Somatostatin, a hypothalamic tetradecapeptide, has been shown to inhibit the release of PRL, GH, and TSH from the pituitary gland. We have characterized specific binding sites for somatostatin on AtT20/D16v cells and demonstrate that somatostatin inhibits stimulated ACTH release by these cells. Equilibrium binding studies with [125I]Tyr1]somatostatin showed the presence of a single class of noninteracting binding sites on AtT20/D16v cells. Half-maximal binding of somatostatin occurred at 1.7 X 10(-9) M, and there were 26,300 binding sites/cell. The binding of [125I]Tyr1]somatostatin was not significantly inhibited by the hypothalamic peptides TRH, LHRH, and substance P. Somatostatin had no consistent effect on basal ACTH secretion by AtT20/D16v cells, but it inhibited ACTH secretion stimulated with either 50 mM KCl or a hypothalamic extract. Half-maximal inhibition occurred with 4 X 10(-10) M somatostatin. TRH, LHRH, and substance P at concentrations of 10(-7) M were without effect. Somatostatin had no effect on either basal or stimulated hormone secretion by GH12C1 or F4C1 cells, two cell strains which lack specific somatostatin-binding sites. A critical concentration of extracellular calcium was required for the stimulation of ACTH secretion in AtT20/D16v cells. No response to 50 mM KCl occurred in the presence of EGTA or cobalt. Increased extracellular calcium overcame the inhibition of stimulated hormone secretion by EGTA, cobalt, and somatostatin. Therefore, we conclude that the inhibition of stimulated ACTH secretion by somatostatin involves the interaction of the peptide with specific binding sites on AtT20/D16v cells and the inhibition of stimulus-elicited calcium influx.

Establishment in culture of a multihormone-secreting cell strain derived from the MtT/F4 rat pituitary tumor.

A clonal cell strain F4C1 has been established from the transplantable rat pituitary tumor MtT/F4 and has been maintained in continuous culture for two years. The cells grow with a population doubling time of 48 hours; the karyotype with a modal number of 39 chromosomes includes a pair of large metacentric marker chromosomes. F4C1 cells in culture produce growth hormone and prolactin but not adrenocorticotropin in contrast to the MtT/F4 tumor which secretes all three hormones in the host rat. The cloned cells lack specific receptors for thyrotropin-releasing hormone and do not respond to this agent with increased prolactin or decreased growth hormone production. Treatment with hydrocortisone results in a small increase in growth hormone and a small decrease in prolactin production. Tumors generated in rats from injected F4C1 cells secrete prolactin and growth hormone but not adrenocorticotropin. The results suggest that growth hormone and prolactin are produced by a single cell type in the MtT/F4 tumor.

Unexpected resistance to x-irradiation in a strain of hybrid mammalian cells.

The radiosensitivities of a strain of mouse fibroblasts (Cl-1D), of rat pituitary cells (GH(1)2C(1)), and of a hybrid between the two (alpha-RST) have been studied. Their mean chromosome numbers were 50, 70, and 111, respectively. The hybrid cells were much more resistent to radiation than either of the parent strains. The range of the D(0) (reciprocal of the slope, and therefore a measure of radiosensitivity) for the linear portion of the survival curves for each cell line was: Cl-1D, 134-142 R; GH(1)2C(1), 154-170 R; and alpha-RST, 248-274 R. There were no significant differences in the magnitude of the shoulder or extrapolation number of the survival curves, nor in the ability of the three cell strains to accumulate and repair sublethal radiation damage. It appears unlikely that the unusual resistance of the hybrid strain is simply related to the increase in chromosome number; more likely, it involves some interaction between the two genomes. The study of somatic cell hybrids may offer further insight into the factors controlling the radiosensitivity of mammalian cells.