Platelet secretion of beta-amyloid is increased in hypercholesterolaemia.

Tissue accumulation of the cytotoxic beta-amyloid peptide (Abeta) occurs in Alzheimer's disease (AD), one possible source being the platelet. AD and cardiovascular disease may share some risk factors, including hypercholesterolaemia which is associated with increased platelet activity. We examined platelet Abeta release under resting and collagen-stimulated conditions in normocholesterolaemic and hypercholesterolaemic individuals. Resting platelet Abeta efflux was greater in hypercholesterolaemics than in normocholesterolaemics. Collagen-stimulated Abeta release was concentration-dependent and increased in hypercholesterolaemics. Resting Abeta release correlated positively with plasma total cholesterol and low-density lipoprotein (LDL) cholesterol, and inversely with platelet count. These data indicate that abnormal platelet Abeta release occurs in hypercholesterolaemia.

Angiotensin II-induced inositol phosphate production in isolated rat zona glomerulosa and fasciculata/reticularis cells.

Angiotensin II (2.5 to 250nM) induced, within 60 sec, a significant increase in [3H]inositol-labeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate in rat zona glomerulosa cells. Neither ACTH (3nM) nor K+ (8.4mM) had any effect, although aldosterone and corticosterone were significantly stimulated by all three agonists (after 30 min incubation). A similar significant dose-dependent increase in the inositol phosphates was observed with angiotensin II in zona fasciculata/reticularis cells after 30 min, but without any effect on corticosterone. In contrast ACTH significantly increased corticosterone with only a small although highly significant increase in inositol trisphosphate and inositol bisphosphate at 0.03nM ACTH. However at the higher dose (3.0nM) only inositol bisphosphate was significantly increased. These results indicate the presence on both zona glomerulosa and zona fasciculata/reticularis cells of AII receptors, which were linked to the formation of the secondary messenger, but only in the zona glomerulosa cells are associated with steroidogenesis.

Effects of alpha-melanocyte-stimulating hormone on the cyclic AMP and phospholipid metabolism of rat adrenocortical cells.

Results on the effects of peptides on the phospholipid metabolism and steroid and cyclic AMP (cAMP) outputs of rat adrenal capsular cells (96% zona glomerulosa, 4% zona fasciculata) were obtained in a series of three batch experiments. Their significance was examined by analysis of variance. Incorporation of [32P] into phosphatidylcholine, phosphatidic acid and phosphatidylinositol was measured. Production of [3H]inositol-1 monophosphate, inositol-1,4 bisphosphate and inositol-1,4,5 tris-phosphate was estimated after prelabelling with [3H]inositol followed by 1 min incubation with a steroidogenic stimulus. Angiotensin II (0.25 nmol/l to 0.25 mumol/l) highly significantly (P less than 0.01) stimulated aldosterone and corticosterone outputs, [32P] incorporation into phosphatidic acid and phosphatidylinositol (but not into phosphatidylcholine) and the production of the three [3H]inositol phosphates. Aldosterone and corticosterone outputs were stimulated by alpha-MSH (above 0.1 nmol/l). However, incorporation of [32P] was not significantly increased until 10 mumol alpha-MSH/l but, unlike with angiotensin II, incorporation into phosphatidylcholine was also then stimulated. Also, the production of the inositol phosphates was not increased significantly (P greater than 0.05) by any dose of alpha-MSH (10 nmol/l, 1 mumol/l and 0.1 mmol/l) used. Therefore, it can be concluded that alpha-MSH does not stimulate phospholipase C in rat zona glomerulosa cells. In further experiments, it was also found that there were significant increases in cAMP as well as in steroid outputs above 1 nmol alpha MSH/l (highly significant above 10 nmol alpha-MSH/l). There were plateaux of the outputs of both steroids and cAMP from 0.1 to 1 mumol alpha-MSH/l. However, there were further increases in steroid and cAMP outputs of the capsular cells at higher doses. Concomitant results on the stimulation of corticosterone output by zona fasciculata-reticularis cells indicate that this additional increase was mostly due to the stimulation of the contaminating zona fasciculata cells. It was also confirmed that alpha-MSH preferentially stimulates steroidogenesis by the zona glomerulosa. However, under our conditions, alpha-MSH highly significantly increased the output of cAMP by both zona fasciculata and glomerulosa cells.

Purification of dispersed rat adrenal zona glomerulosa cells by Percoll density gradient centrifugation and the isolation of a population of cells highly responsive to adrenocorticotrophin.

Percoll density gradient centrifugation is a simple, inexpensive and convenient method to eliminate contaminating zona fasciculata (ZF) cells from unpurified rat adrenal capsular glomerulosa (ZG) cell preparations (with less than 0.1% ZF cells in the final cell preparation). Basal steroid (aldosterone and corticosterone) output by the purified (PG) cells was unchanged. These purified cells, although free from ZF contamination, were more highly responsive than expected to ACTH (3 nmol/l). When PG cells were further separated by Sephadex column filtration, the filtered PG cells exhibited the steroidogenic response of ZG cells purified by unit gravity sedimentation and Sephadex column filtration, i.e. reduced basal steroid output and an ACTH response reduced to that stimulated by K+ (8.4 mmol/l). Although the cells retained in the column resembled the filtered PG cells ultrastructurally, they showed unchanged basal steroid output and a high ACTH response with increased late-pathway activity (the conversion of corticosterone to aldosterone). By combining Percoll density gradient centrifugation and Sephadex column filtration we have a method for the isolation and study of both the high- and low-response rat ZG cells which are free from ZF contamination.

The mechanism of the effect of K+ on the steroidogenesis of rat zona glomerulosa cells of the adrenal cortex: role of cyclic AMP.

The effects of various concentrations of extracellular K+ (3.6-13 mM) on the steroid (corticosterone and aldosterone) and cyclic AMP outputs of capsular cells (95% zona glomerulosa) of the rat adrenal cortex were studied at different concentrations of extracellular Ca2+. Small amounts of EGTA (50 microM) were added to reduce the free Ca2+ concentrations effectively to zero at the lowest possible total Ca2+ concentration. At a total extracellular concentration of 2.5 mM Ca2+, in 27 experiments the mean values of the steroid and cAMP outputs showed a maximum at 8.4 mM K+. The increase in steroid and cAMP outputs at 5.9, 8.4 and 13 mM K+ compared with that at 3.6 mM were highly significant (p less than 0.01). The overall correlation of either corticosterone or aldosterone with cAMP outputs was also highly significant and was even better from 3.6 to 8.4 mM K+. Lowering the effective free concentration of Ca2+ to zero decreased the steroid and cAMP outputs significantly at all K+ concentrations, and no output was then significantly higher than at 3.6 mM. With the pooled data on outputs at all total Ca2+ (2.5, 0.5, 0.25, 0.10, 0.05 and 0.0 mM) and K+ (3.6, 5.9, 8.4 and 13 mM) concentrations, the correlation of either steroid with cAMP outputs was highly significant (but again optimally from 3.6 to 8.4 mM K+). Nifedipine (10(-6) to 10(-4) M) was added to the incubations with the aim of specifically inhibiting Ca2+ influx at total extracellular Ca2+ concentrations of 2.5, 1.25 and 0.25 mM and with the usual K+ concentrations. The cAMP outputs were reduced at all K+ concentrations above 3.6 mM K+. The effect was highly significant at 10(-4) M nifedipine and a total Ca2+ of 1.25 mM, which with the incubation conditions used, corresponds to the free Ca2+ concentrations in vivo. These results indicate that cAMP plays a significant role in the stimulation of steroid output by K+ particularly between 3.6 and 8.4 mM K+. In this range of K+ concentrations the stimulation of cAMP seems to be controlled by increases in Ca2+ influx. The correlation of steroid and cAMP output at the higher K+ concentrations (between 8.4 and 13 mM K) and at the various total Ca2+ concentrations is less significant. Also, with all concentrations of added nifedipine there is an 'anomalous' increase in steroid output at 13 mM K+ and at total Ca2+ concentrations of 2.5 and 1.25 mM. However, at the same K+ concentrations and at 0.25 mM Ca2+, nifedipine decreases steroid outputs.(ABSTRACT TRUNCATED AT 400 WORDS)

Steroid biosynthesis by zona fasciculata and zona reticularis cells purified from the mammalian adrenal cortex.

Steroidogenesis by zona reticularis (ZR) and zona fasciculata (ZF) cells from guinea-pig, macaque and human adrenals has been examined to establish the relative importance of the two cell types in the production of adrenal androgens. Microdissection and equilibrium density sedimentation of enzymically dispersed cells on Percoll gradients have been used to separate the two cell types with varying degrees of purity. Outputs of androstenedione and cortisol were measured for all the species studied and dehydroepiandrosterone sulphate (DHAS) for human adrenal cells. In all three species, the ZF cells produced more cortisol than the ZR cells. This production was stimulated to a greater extent in the ZF than the ZR cells in the guinea-pig, but to a similar extent in the macaque and human cells. Outputs of androstenedione were greater for ZF cells than ZR cells in the guinea-pig both basally and in response to an ACTH stimulus but basal production of this androgen was slightly greater from the ZR than the ZF in tissue incubations (1.6-fold), although cortisol output was still higher for ZF tissue by a factor of 2. Examination of androstenedione/cortisol ratios for all the species, both tissue and cells and in the presence or absence of ACTH, demonstrated clearly the inherent bias for ZR cells towards androgen synthesis by the fact that this ratio was always higher for ZR than for ZF cells or tissue. The most marked difference in this ratio was found in the human cells comparing the purest ZR and the purest ZF cell preparations even though the highest estimated purity of either cell type was only 80%. The difference in the ratio DHAS/cortisol was even greater comparing these human ZR and ZF cell preparations, ranging from 18- to 44-fold basally and in response to ACTH. The preliminary experiment on human adrenal cells indicates that the ZR cells are much more important than the ZF cells in the production of androgens even in the presence of ACTH, in contrast to the other species studied.

Preparation and steroidogenic properties of purified zona fasciculata and zona reticularis cells from the guinea-pig adrenal gland.

Mixtures of zona fasciculata (ZF) and zona reticularis (ZR) cells, obtained by enzyme dispersion of decapsulated guinea-pig adrenal glands, were separated either by unit gravity sedimentation or by equilibrium density sedimentation. There was no evidence of deleterious effects on ultrastructural integrity or the ability of cells to respond to (1-24)ACTH (Synacthen) after either separation technique. Unit gravity sedimentation gave one fraction in which 90% of the cells were from the ZR and another fraction in which 70% of the cells were from the ZF. Equilibrium density sedimentation of cell mixtures on Percoll gradients gave fractions containing either 90% pure ZR or 95% pure ZF cells. Cortisol, 11-deoxycortisol, corticosterone, deoxycorticosterone, 11 beta-hydroxyandrostenedione and androstenedione were all formed from [14C]pregnenolone on incubation with purified preparations of both types of cell. No product was seen to be unique to either cell type although ZR cells appeared deficient in 11 beta-hydroxylase activity relative to ZF cells. The ratio of androstenedione to cortisol (formed either from labelled pregnenolone or from endogenous precursors) was higher for ZR cells than for ZF cells. When the purer cells obtained by equilibrium density sedimentation were studied, it was found that (1-24)ACTH stimulated greater steroid production (both androstenedione and cortisol) by the ZF cells compared with the ZR cells.

Further studies on the stimulation of rat adrenal capsular cells: four types of response.

Preparations of capsular rat adrenal cells consisting mainly of zona glomerulosa with less than 5% zona fasciculata contamination are described. The responses of the aldosterone and corticosterone outputs of these preparations to various stimuli were of four types. (1) Variations in K+ concentration gave a maximum aldosterone response at 5.9-8.4 mM-K+, about sixfold greater than the control output at 3.6 mmol/l. At higher K+ concentrations such as 13 mmol/l, the response decreased. (2) Serotonin (at a concentration of about 10(-4) mol/l) gave only a slightly lower maximal aldosterone response than did K+ but this did not decrease significantly at higher concentrations. Serotonin gave significant steroidogenic response at 10(-8) mol/l. (3) [Asp1,Val5]-Angiotensin II (10(-10) mol/l) with 3.6 mM-K+ gave a significant response and a constant maximal response at 2.5 x 10(-8) mol/l. This maximum response was about half that found for both aldosterone and corticosterone when stimulated maximally by K+ or serotonin: [des-Asp1,Ile5]- and [des-Asp1,Val5]-angiotensin II (angiotensin III) gave similar response characteristics but had a lower potency in this cell preparation. The initial maximum response could be further increased at a higher concentration (from 2.5 x 10(-5) mol/l) of a preparation of [Asn1,Val5]-amide angiotensin II (Hypertensin-Ciba) and might eventually be greater than with K+. This additional response was, to a major extent, due to stimulation of the contaminating zona fasciculata cells and was not seen with high concentrations of the free acid, angiotensin II. It was also not seen in two experiments with pure [Asn1]-amide angiotensin II and therefore it could have been due to some impurity in Hypertensin-Ciba. (4) Adrenocorticotrophin (Synacthen) at 3 x 10(-11) mol/l gave a significant steroidogenic response. Higher concentrations (3 x 10(-10) to 7.5 x 10(-9) mol/l) gave no constant maximum but the response could be much greater than for other stimuli such as K+, serotonin and [Asp1]-angiotensin II. This additional response was again due to steroid precursors, e.g. deoxycorticosterone and corticosterone from contaminating zona fasciculata cells. Similar results were obtained with ACTH (ACTHAR) in three experiments. Threshold sensitivity (a significant increase in steroidogenesis) for ACTH (Synacthen) was, in two experiments, greater for zona fasciculata-reticularis cells (3 x 10(-12) mol/l) than for zona glomerulosa cells (3 x 10(-11) mol/l). The data show that aldosterone output was approximately a function of the square of the corresponding corticosterone value. Specific effects on this pathway can be shown by values of aldosterone/corticosterone2 greater than one. Of all stimuli used, only K+ concentrations of 5.3, 5.9 and 13 mmol/l gave such effects. However, because of several considerations, only positive results with other stimuli may be meaningful. Calculation of this parameter might be useful as a screening test in bioassays for substances with aldosterone-stimulating activity.

Cyclic AMP levels in purified rat adrenal zonae fasciculata and reticularis cells and the effect of adrenocorticotrophic hormone.

Cyclic AMP levels were measured in combined cells and supernatant fraction from incubations of dispersed rat adrenal zona fasciculata and zona reticularis cell preparations purified by unit gravity sedimentation. These measurements were correlated with deoxycorticosterone (DOC) and corticosterone outputs from the cells in the presence or absence of ACTH. Similar measurements of cyclic AMP outputs were made for unpurified dispersed, decapsulated rat adrenal cell preparations and they were found to correspond to previously reported measurements made by other workers on such preparations. The response of the purest zona reticularis cells to ACTH in terms of cyclic AMP output was 28-fold lower than that of the purest zona fasciculata cells (compared with a fivefold lower DOC output and a 20-fold lower corticosterone output) and the response to ACTH of the mixed-cell preparations was related to the number of zona fasciculata cells in the preparation, i.e. the greater the proportion of zona fasciculata cells in the preparation the greater the response in terms of both outputs of cyclic AMP and of either of the two steroids measured. This correlation is in accordance with the theory that cyclic AMP may be the secondary messenger for both zona fasciculata and zona reticularis cells of the rat adrenal cortex in mediating the response to an ACTH stimulus.

Properties of rat adrenal zona reticularis cells: production and stimulation of certain steroids.

The outputs of corticosterone, deoxycorticosterone and androstenedione from dispersed, purified rat adrenal zona reticularis and zona fasciculata cells have been measured by radioimmunoassay. Preferential production of deoxycorticosterone by zona reticularis cells was demonstrated by their higher basal deoxycorticosterone : corticosterone ratio when compared with zona fasciculata cells. Adrencorticotrophin (ACTH) stimulated corticosterone output by all cell pools prepared by unit gravity (1 g) sedimentation, zona fasciculata cells being stimulated 130-fold compared with 20-fold for the zona reticularis cells in relation to their basal corticosterone output. In every cell pool, ACTH stimulated the output corticosterone more than it stimulated the output of deoxycorticosterone. In parallel cell preparations, it was shown that ACTH increased the conversion of tracer amounts of radioactive deoxycorticosterone to corticosterone and decreased the conversion of radioactive corticosterone to 11-dehydrocorticosterone. Adrenocorticotrophin did not increase the conversion of radioactive deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone). It is unlikely therefore that ACTH stimulates 11 beta-hydroxylation. Data indicate that the ratio of deoxycorticosterone to total 11-oxygenated steroids (corticosterone + 11-dehydrocorticosterone) is characteristic for each cell type, and that this ratio will be relatively independent of ACTH stimulation or the amount of pregnenolone substrate available. Basal androstenedione outputs were similar for both types of cell, and ACTH stimulation was very small, being slightly greater for zona fasciculata than for zona reticularis cells. The contribution of the zona reticularis cells to the basal output of any steroid by the cells of the inner two zones of the adrenal cortex of the rat was relatively small (20% for deoxycorticosterone and 10% for corticosterone) and was even less after stimulation by ACTH. Unless a specific stimulus can be found, therefore, a significant role for the zona reticularis cannot yet be established.

Properties of rat adrenal zona reticularis cells: preparation by gravitational sedimentation.

An enriched fraction of zona reticularis cells was obtained by unit gravity sedimentation of decapsulated adrenal glands from female rats. From light microscopic and ultrastructural studies of the whole gland and the isolated cell fractions, the zona reticularis cells of the adrenal gland can be classified mainly on the bases of size, position and mitochondrial morphology. This cell population consists of two types of cell, the 'true' zoma reticularis cells (Type I, modal diameter 9 micrometer), which usually constitute 90% of the isolated reticularis fraction and 80% of the intact reticularis tissue, and cells (Type II, modal diameter 13 micrometer) with fasciculata-like properties (rich in lipid and spherical mitochondria with vesicular cristae). Staining of the cell preparation for 3beta-hydroxysteroid dehydrogenase activity also demonstrates the existence of two types of cell in the zona reticularis. The zona reticularis cell fraction, like the zona fasciculata cell fraction, was capable of producing the subsequent steroids from radioactive pregnenolone: corticosterone, deoxycorticosterone, 18-hydroxydeoxycorticosterone, 11-dehydrocorticosterone, progesterone and androstenedione. However, the pattern of steroid production differed markedly between the zona reticularis and zona fasciculata cells, particularly with respect to the production of deoxycorticosterone and corticosterone (and its correlated steroids, 11-dehydrocorticosterone and 18-hydroxydeoxycorticosterone). When R (the ratio of deoxycorticosterone : corticosterone plus 11-dehydrocorticosterone) for the purest preparation of reticularis cells was compared with R for the corresponding preparation of fasciculata cells, the normalized ratio was found to be 6.4, 16.4 and 20.1 in three experiments. The pattern of production of androstenedione per cell was similar in the reticularis and fasciculata cell fractions. The exact mechanism for the altered pattern of steroid metabolism remains to be elucidated. However, these results establish that the corticosteroids produced by the cells of the zona reticularis may be quantitatively, if not qualitatively, different from those produced by the zona fasciculata cells.