Utility of unbound plasma drug levels and P-glycoprotein transport data in prediction of central nervous system exposure.

1. Drug concentrations in cerebrospinal fluid have been assumed to be a natural surrogate for total drug exposures in the central nervous system. The present communication reports a data set from a study of 30 compounds in mice. An attempt was made to correlate cerebrospinal fluid and unbound plasma drug concentrations via incorporation of in vitro P-glycoprotein (Pgp)-mediated transport data. 2. Pgp-deficient (Pgp -/-) and wild-type mice were dosed with compounds of interest by oral gavage (orally) at 5 mg kg(-1). Plasma and cerebrospinal fluid samples were collected at 1 h post-dosing, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for drug concentrations. Mouse and human Pgp-mediated transport were evaluated in vitro by a bi-directional (B to A and A to B) transport assay using LLC-PK1 cells expressing mouse (mdr1a) and human (MDR1) forms of Pgp, respectively. 3. Compounds with B to A/A to B transport ratios < 2 were defined as non-substrates of Pgp, whereas those exhibiting B to A/A to B transport ratios > or =2 were considered Pgp substrates. Plasma protein binding was also determined in vitro via equilibrium dialysis. Of the 30 compounds, 13 were identified to be mouse Pgp substrates, all of which were also human Pgp substrates, demonstrating a good agreement between mouse and human data. 4. In Pgp wild-type mice, the unbound plasma and cerebrospinal fluid concentrations of the non-Pgp substrates correlated well, with a regression slope of approximately 1.0. A similar relationship existed for Pgp substrates in Pgp -/- mice. On the other hand, an improved correlation of cerebrospinal fluid and systemic exposures of the Pgp substrates in Pgp wild-type mice was observed when the unbound plasma concentrations were normalized to the corresponding B to A/A to B transport ratios. 5. These results reinforce the premise that a combined use of unbound plasma drug concentrations and in vitro Pgp transport data may be of value for the estimation of central nervous system exposures.

Differentiating the mechanisms of antiresorptive action of nitrogen containing bisphosphonates.

Bisphosphonates (BPS) inhibit bone resorption and are divided into two classes according to their chemical structure and mechanism of action: nonnitrogen containing BPS such as etidronate and clodronate that are of low potency and inhibit osteoclast function via metabolism into toxic ATP-metabolites and nitrogen-containing BPS (NBPS), such as alendronate and risedronate that inhibit the enzyme of the mevalonate biosynthetic pathway farnesyl pyrophosphate synthase (FPPS), resulting in inhibition of the prenylation of small GTP-binding proteins in osteoclasts and disruption of their cytoskeleton. Previously, studies in various cell types suggested, however, that pamidronate functions by mechanism(s) additional or independent of the mevalonate pathway. To examine if such mechanism(s) are also involved in the action of NBPS on osteoclastic bone resorption, we examined the action of alkyl and heterocyclic NBPS with close structural homology on FPPS/isopentenyl pyrophosphate isomerase (IPPI) activity, on osteoclastic resorption, and on reversibility of this effect with GGOH. As expected, both pamidronate and alendronate suppressed bone resorption and FPPS/IPPI activity, the latter with greater potency than the first. Surprisingly, however, unlike alendronate, the antiresorptive effect of pamidronate was only partially reversible with GGOH, indicating the involvement of mechanism(s) of action additional to that of suppression of FPPS. Comparable results were obtained with the heterocyclic NBP NE-21650, a structural analog of risedronate. Thus, despite an effect on FPPS, the actions on bone resorption of some NBPS may involve mechanisms additional to suppression of FPPS. These findings may lead to identification of additional pathways that are important for bone resorption and may help to differentiate among members of the NBP class which are currently distinguished only according to their potency to inhibit bone resorption.

Role of major lifetime stressors in patients' and spouses' reactions to cancer.

This study examined the role of number of major lifetime stressors (e.g., rape, abuse), and the perceived resolution of those stressors, in cancer patients' (n = 54) and spouses' (n = 30) appraisals and current mood. We hypothesized that a high number of lifetime stressors, and low resolution ratings, would be associated with more distress and more negative appraisals of the cancer. Hierarchical regression analyses showed that number of lifetime stressors was a positive predictor of patients' ratings of the cancer's threat, and a positive predictor of their spouses' anger. Mean resolution ratings were a significant positive predictor of spouses' positive affect. The findings suggest that experience with previous stressors affects an individual's reactions to cancer.

The effects of altitude on heavy-duty diesel truck on-road emissions.

On-road measurements of carbon monoxide, hydrocarbons, and nitric oxide from 5772 heavy-duty diesel trucks at five locations in the United States and Europe show slightly increasing emissions with increasing altitude. The result for nitric oxide showed a statistically significant increase of 4.1 +/- 1 gNO/kg of fuel consumed/km increase in altitude. The increases for CO and HC were also statistically significant.

LC/MS/MS quantitation of an anti-cancer drug in human plasma using a solid-phase extraction workstation: application to population pharmacokinetics.

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate an anti-cancer drug in human plasma was validated. The method has proven suitable for routine quantitation of the experimental anti-cancer compound at concentrations from 1 to 400 ng/ml. Retention times of the compound and internal standard (compounds I and II, respectively) were 1.8 and 2.1 min, respectively. No interfering endogenous peaks were observed throughout the validation process. Precision estimates for this approach were typically less than 5% relative standard deviation (RSD) across the calibration range. Other validation parameters studied included specificity, system reproducibility, limit of quantitation, accuracy, linear range, and stability of the compound and internal standard in plasma and injection solvent. This method was used to quantify drug for population pharmacokinetic studies.

Differential effect of simvastatin on various signal transduction intermediates in cultured human smooth muscle cells.

The underlying mechanism of the antiproliferative effect of S (simvastatin), a HMG-CoA reductase inhibitor, in vascular smooth muscle cells (SMC) is still poorly understood. In the present study, we used synchronized human SMC, isolated from left interior mammary artery, as an in vitro model to test the effects of S on platelet-derived growth factor (PDGF)-induced DNA synthesis, extracellular-regulated kinase 1/2 (ERK1/2), p38/stress-activated protein kinase 2 (SAPK2), RhoA and Rac1 activation. ERK1/2 phosphorylation was triggered within 2 min of PDGF stimulation (early G1 phase) and was blocked by PD98059, a specific inhibitor of the ERK1/2 pathway, which also strongly inhibited PDGF-induced DNA synthesis (IC(50) = 10 micromol/L). PDGF quickly induced p38 phosphorylation (early G1 phase) and SB203580, a specific inhibitor of the p38/SAPK2 pathway, also blocked PDGF-induced DNA synthesis (IC(50) = 0.3 micromol/L). Translocation to the plasma membrane of small GTPases, such as RhoA and Rac1, could not be detected within 15 min of stimulation with PDGF or lysophosphatidic acid (LPA) (early G1 phase), but occurred after 24 hr of PDGF stimulation (late G1/S phase). S inhibited PDGF-induced DNA synthesis (IC(50) = 3.5 micromol/L), and this effect was dependent on intracellular mevalonate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate availability. The critical time period for the reversal of the S effect by mevalonate comprised both the early and late G1 phase of the SMC cycle. PDGF-induced ERK1/2 phosphorylation and PDGF-induced p38 phosphorylation were not markedly affected by S during the whole G1 phase. However, S treatment blocked the PDGF- and LPA-induced membrane translocation of RhoA that occurred during the late G1/S phase. In the case of Rac1, the same process was also inhibited by S treatment. We concluded from these results that, in SMC, the early events associated with ERK1/2 and p38 signal transduction pathways, recruited for PDGF-mediated DNA synthesis, were insensitive to S action, whereas the mevalonate-dependent, posttranslational modification of RhoA and Rac1 molecules, required for PDGF-induced membrane translocation, was blocked by this drug. These results suggest that the antiproliferative effect of S can be explained not only by the blockage of RhoA-mediated signaling events but also by Rac1-mediated signaling events.

Inhibitors of prenylation of Ras and other G-proteins and their application as therapeutics.

Anchoring of small G-proteins to cellular membranes via a covalently bound lipophylic prenyl group is essential for the functioning of these proteins. For example, the farnesylation of Ras by the action of the enzyme protein:farnesyl transferase (PFT) is pivotal for its signalling function in cell growth and differentiation. The development of inhibitors of PFT was triggered by the role of mutated Ras in certain types of cancer and by the observation that non-farnesylated Ras is inactive. Besides the screening of existing compounds for PFT inhibition, rational drug design has also led to new inhibitors. Our research is in the field of atherosclerosis and concerns the development of inhibitors of the growth of vascular smooth muscle cells. The latter process gives rise to reocclusion of the coronary artery (restenosis) after balloon angioplasty. We and others have developed several analogues of the two substrates of PFT, i.e. farnesyl pyrophosphate (FPP) and the so-called CAAX peptide consensus sequence, which were tested in vitro for the inhibition of PFT and of other enzymes involved in protein prenylation, such as protein:geranylgeranyl transferase-1 (PGGT-1). The FPP analogue TR006, a strong inhibitor of PFT (IC(50) of 67 nM), blocked the proliferation of cultured human smooth muscle cells and inhibited platelet-derived growth factor- and basic fibroblast growth factor-induced DNA synthesis. Similar but more highly charged compounds failed in this respect, probably because of an impaired uptake in the cells. Less charged derivatives were designed to circumvent this problem. The effect on the GF-induced activation of intermediates in signal transduction pathways was investigated in order to gain insight into the mechanism of action within the cells. TR006 decreased the bFGF activation of extracellular signal-regulated kinase 1 (ERK1), suggesting its involvement in inhibiting Ras activity. Although other analogues inhibited DNA synthesis, they affected neither ERK1 activation nor p38/stress-activated protein kinase 2 or Jun N-terminal kinase 1 activation. Since some of these compounds were also shown to be inhibitors of in vitro PGGT-1 activity, the geranylgeranylation of other G-proteins may be decreased by these compounds. Rho seems to be a good candidate as a target for inhibitors of PGGT-1. This uncertainty as to the mechanism of action within non-transformed as well as transformed cells applies to all prenylation inhibitors, but is not holding back their further development as drugs. Their current and possible future application as therapeutics in cancer, restenosis, angiogenesis, and osteoporosis is briefly discussed.

Equally potent inhibitors of cholesterol synthesis in human hepatocytes have distinguishable effects on different cytochrome P450 enzymes.

Six 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (the present cholesterol-lowering drugs known as statins), lovastatin (L), simvastatin (S), pravastatin (P), fluvastatin (F), atorvastatin (A) and cerivastatin (C) are shown to be potent inhibitors of cholesterol synthesis in human hepatocytes, the target tissue for these drugs in man. All six inhibited in the nM range (IC(50) values: 0.2-8.0 nM). As daily used cholesterol-lowering drugs they are likely coadministered with other drugs. While several cytochrome P450 (CYP) enzymes are involved in drug metabolism in the liver and thus play an important role in drug-drug interaction it was investigated which of these enzymes are influenced by the active forms of the six statins. These enzyme activities were studied in human liver microsomal preparations, and in simian and human hepatocytes in primary culture. The following CYP reactions were used: nifedipine aromatization (CYP3A4), testosterone 6beta-hydroxylation (CYP3A4), tolbutamide methylhydroxylation (CYP2C9), S-mephenytoin 4-hydroxylation (CYP2C19), bufuralol 1'-hydroxylation (CYP2D6), aniline 4-hydroxylation (CYP2E1), coumarin 7-hydroxylation (CYP2A6) and 7-ethoxyresorufin O-dealkylation (CYP1A1/2). In the human liver microsomes the statins (concentrations up to 400 microM) did not influence the CYP1A1/2 activity and hardly the CYP2A6 and CYP2E1 activities. Except P, the other five statins were stronger inhibitors of the CYP2C19 activity with IC(50) values around 200 microM and the same holds for the effect of A, C and F on the CYP2D6 activity. L and S were weaker inhibitors of the latter enzyme activity, whereas P did not influence both activities. About the same was observed for the statin effect on CYP2C9 activity, except that F was a strong inhibitor of this activity (IC(50) value: 4 microM). Using the assay of testosterone 6beta-hydroxylation the CYP3A4 activity was decreased by L, S and F with IC(50) values of about 200 microM and a little more by C and A (IC(50) around 100 microM). P had hardly an effect on this activity. To a somewhat less extent the same trend was seen when CYP3A4 activity was measured using nifedipine as substrate. The inhibitory effects observed in microsomes were verified in suspension culture of freshly isolated hepatocytes from Cynomolgus monkey (as a readily available model) and of human hepatocytes. In general the same trends were seen as in the human microsomes, except that in some cases the inhibition of the CYP activity was less, possibly by the induction of the particular CYP enzyme by incubation of the cells with a particular statin. F remained a strong inhibitor of CYP2C9 activity in human and monkey hepatocytes. A induced the CYP2C9 in monkey hepatocytes but was an inhibitor of the CYP2C9 in human hepatocytes. A, S, L and C were moderate inhibitors in both cellular systems of CYP3A4. P was not affecting any of the CYP activities in the three systems studied. It is concluded that different CYP enzymes interact with different statins and therefore differences in between these drugs are to be expected when drug-drug interaction is considered.

Pyrimidine dimer formation as a probe of nucleosome core and linker structure in situ.

The photoinduced dimerization of adjacent pyrimidines in DNA is influenced in predictable ways by DNA conformation. A method is described for determining patterns of pyrimidine dimer formation under conditions in which the chromatin is minimally perturbed. The relation of such patterns to the conformation of nucleosomal core DNA and linker DNA, as well as the interaction of histone H1 with nucleosomal DNA, is presented. Such data indicate that sharp bends in the path of DNA seen in crystals of isolated nucleosome core particles are also present in intact chromatin. They also indicate that most of the linker has very little curvature except for a small bend at its junction with the nucleosome core. The linker path inferred from such experiments supports models in which the chromatin fiber consists of a zigzag chain of nucleosomes.

The role of neuroticism in daily stress and coping.

The authors examined the influence of neuroticism (N) on the occurrence of different types of daily events, primary and secondary appraisals of those events, use of specific coping strategies, and end-of-day negative mood. College students completed questionnaires at the end of every day for 14 consecutive days. When reporting their most stressful event of each day, high-N individuals, compared with low-N individuals, reported more interpersonal stressors and had more negative primary and secondary appraisals and reacted with more distress in response to increasingly negative primary and secondary appraisals. Compared with low-N individuals, high-N individuals used less-adaptive coping strategies (e.g., hostile reaction) and reacted with more distress in response to some types of coping strategies. The appraisal findings, in particular, help to explain the chronic negative affectivity associated with neuroticism.

Inhibition of human smooth muscle cell proliferation in culture by farnesyl pyrophosphate analogues, inhibitors of in vitro protein: farnesyl transferase.

In this study, it was investigated whether and how inhibitors of protein:farnesyl transferase (PFT) can inhibit the proliferation of human smooth muscle cells (HSMC) in culture. Several farnesyl pyrophosphate (FPP) analogues were synthesized and tested in vitro for their specificity in inhibiting squalene synthase (SS), PFT, or protein:geranylgeranyl transferase-1 (PGGT-1) activities (the latter was determined using a newly designed assay). One of these compounds appeared to be a strong PFT inhibitor (IC50 value: 340 nM) and a weak inhibitor in the other two enzyme assays. This compound (designated as TR006) inhibited the farnesylation of Ras in a Ha-ras transfected cell line (Cohen et al., Biochem. Phamacol. 49: 839-845, 1995) and concomitantly slowed down the growth of these cells. Twenty-five microM of TR006 inhibited the proliferation of HSMC isolated from left internal mammary artery, as measured by counting the cells over a period of three cell cycles (10 days). A structurally related compound (TR007), a specific SS inhibitor, did not influence HSMC proliferation under the same conditions. The inhibition by TR006 was concentration-dependent. In HSMC, synchronized by serum depletion, platelet-derived growth factor (PDGF) or basic fibroblast growth factor (bFGF)-induced DNA synthesis was decreased by a 29-hr pretreatment with 100 microM of TR006, indicating that this inhibitor acted in an early phase of the cell cycle, probably by preventing protein isoprenylation. Some other FPP analogues with comparable IC50 values in the in vitro PFT assay were also able to decrease bFGF-induced DNA synthesis without affecting cell viability. A more negatively charged member of this group, TR018, did not influence the growth factor-induced DNA synthesis, probably due to an impaired uptake into the cells. However, the pivaloyloxomethyl derivative of this compound, which is uncharged, and is thought to be converted into TR018 within the cells, showed a strong decrease in bFGF-induced DNA synthesis in HSMC. These data suggest that the compounds investigated may be developed further for treatment of conditions in which undesirable proliferation of smooth muscle cells plays an important role.

An Assessment of Ethnocultural Beliefs Regarding the Causes of Birth Defects and Genetic Disorders.

With the expansion of genetic services to various cultural groups, genetic counselors encounter clients who hold diverse beliefs inscribed by their culture about health conditions. Thus, clients may attribute the cause of a birth defect or genetic condition to a culturally-based health belief. This present study was conducted as a pilot study in order to assess the beliefs about the causes of birth defects and genetic disorders held by women of different ethnocultural backgrounds. This study proposed that women who do not have a family history of a disorder will differ in their knowledge about the cause of a birth defect or genetic disorder compared to women who have an affected child. In addition, this study determined to what extent culturally-based health beliefs are attributed to being the cause of a birth defect or genetic disorder in the 1990s.

Urodynamic risk factors for renal dysfunction in men with obstructive and nonobstructive voiding dysfunction.

PURPOSE: Urodynamic investigation of men with lower urinary tract symptoms, usually attributed to benign prostatic hyperplasia, often reveals bladder outlet obstruction, detrusor instability and/or diminished vesical compliance. We investigated whether these urodynamic abnormalities alone or in combination contribute to renal dysfunction. MATERIALS AND METHODS: A total of 161 men with lower urinary tract symptoms was evaluated by urodynamics, and outlet obstruction, detrusor instability and decreased compliance (30 ml./cm. water or less) were noted. Serum blood urea nitrogen (BUN) and creatinine were measured. Cases were categorized according to the urodynamic diagnosis. Mean values of serum BUN and creatinine as well as the incidence of elevated BUN and creatinine were compared among groups. RESULTS: Of the cohort 54 men (34%) had elevated BUN and 19 (12%) had elevated serum creatinine. No significant correlation was found between the degree of obstruction and BUN or creatinine level. Mean serum BUN and creatinine, and the incidence of abnormal laboratory tests did not significantly differ among those with outlet obstruction, detrusor instability, both conditions or neither condition. However, in patients with outlet obstruction and detrusor instability there was a significantly increased incidence of azotemia in the subgroup with diminished compliance (78%) versus the subgroup with normal compliance (36%). CONCLUSIONS: In men with voiding dysfunction of a nonneurogenic etiology outlet obstruction with or without detrusor instability does not appear to be a risk factor for elevated BUN and creatinine. However, when decreased bladder compliance is associated with a combination of outlet obstruction and detrusor instability, this risk is substantially increased.

Inhibition of proliferation of human smooth muscle cells by various HMG-CoA reductase inhibitors; comparison with other human cell types.

The effects of 6 HMG-CoA reductase inhibitors: pravastatin, lovastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin were analyzed in cultured human smooth muscle cells, fibroblasts, endothelial cells and myoblasts. In vascular smooth muscle cells, pravastatin was a much weaker inhibitor of cholesterol synthesis than the 5 other drugs which displayed equally strong inhibitory potency. The anti-proliferative effects of these 6 drugs were analyzed by measuring cell number and mitochondrial dehydrogenase activity (MTT assay) after 3 days of incubation. IC25 values for inhibition of proliferation were very similar among the 4 cell types and were in the following order of magnitude: pravastatin << lovastatin = simvastatin = atorvastatin = fluvastatin << cerivastatin. Only in the case of pravastatin was proliferation inhibited at lower concentration in smooth muscle cells than in the other cell types. Proliferation was also assessed by measuring DNA synthesis in these cells. A 3 day-incubation with 1 microM of pravastatin had no effect on this parameter in all 4 cell types. However, 1 microM of simvastatin or lovastatin caused either an inhibition (in smooth muscle cells and endothelial cells) or stimulation (in fibroblasts) of this process. The effects of simvastatin on cell number, mitochondrial dehydrogenase activity and DNA synthesis were counteracted by simultaneous mevalonate addition. Simvastatin treatment was also associated with a change in the post-translational modification of the ras protein in smooth muscle cells, probably by inhibition of its farnesylation. Moreover, simvastatin treatment blocked the PDGF and bFGF-induced DNA synthesis in synchronized smooth muscle cells, whereas it does not affect the fetal calf serum-induced DNA synthesis in synchronized fibroblasts, suggesting that simvastatin blocks various steps of the cell cycle and that this effect depends on the cell type and the growth signalling pathway activated.

Control beliefs and faith as stress moderators for Korean American versus Caucasian American protestants.

Examined relationships among negative life events, four locus of control attributions (Internality, Powerful Others, Chance and God Control), and psychological distress for Korean American versus Caucasian American Protestants. Negative events and Powerful Others beliefs were positively related to distress, whereas Internality was negatively related to distress. Ethnicity and God Control interacted: The relationship between God Control beliefs and anxiety was negative for Caucasians but positive for Koreans. Three-way interactions (Ethnicity x Locus of Control x Negative Events) also emerged. As Caucasians' Powerful Others beliefs increased, the positive relationship between negative events and depression became stronger; Koreans' Powerful Others beliefs had no such effect. As Caucasians' God Control beliefs increased, the negative event-depression relationship changed from positive to negative; the reverse was true for Koreans. Findings support the value of assessing ethnoculture and religiousness in stressful life events research.

Growth failure, encephalopathy, and endocrine dysfunctions in two siblings, one with 5-oxoprolinase deficiency.

UNLABELLED: Two female siblings, born to consanguineous parents, presented with a similar phenotype characterized by severe growth and developmental failure, dysmorphic features, thyroid and gonadal dysfunction, autistic traits and hand stereotypes resembling Rett syndrome. In the elder patient, analysis of urinary organic acids disclosed a very high excretion of 5-oxoproline (4.2 to 8.1 mol/mol creatinine) and enzyme assays of leucocyte extracts revealed a profound deficiency of 5-oxoprolinase. However, normal urinary organic acid profiles were found in the younger child. In view of their distinct dysmorphic features and severe growth deficiency, these siblings cannot be considered as Rett Syndrome variants. The Dubowitz and carbohydrate-deficient glycoprotein syndromes were also excluded clinically and biochemically respectively. We conclude that these patients suffer from a hitherto undescribed autosomal recessive disorder, unrelated to the 5-oxoprolinase deficiency of the elder sib. CONCLUSION: The present findings give evidence that 5-oxoprolinase deficiency is not associated with a distinct morbid phenotype.

Action of lovastatin, simvastatin, and pravastatin on sterol synthesis and their antiproliferative effect in cultured myoblasts from human striated muscle.

Lovastatin, simvastatin, and pravastatin are fairly strong inhibitors of sterol synthesis in human myoblasts in culture. Lovastatin and simvastatin have IC50 values of 19 +/- 6 nM and 4.0 +/- 2.3 nM, respectively. Pravastatin is a weaker inhibitor of sterol synthesis (IC50 value of 110 +/- 38 nM). Through inhibition of mevalonate production, these compounds have a distinct inhibiting effect on cell proliferation. Because proliferation of myoblasts is important in the repair of damaged skeletal muscle, experiments were performed to investigate the effect of lovastatin, simvastatin, and pravastatin on cell proliferation and cell viability. The more potent inhibitors of sterol synthesis, lovastatin, and simvastatin, were able to inhibit the proliferation of these cells during 3 days of incubation with drug concentrations of 1 microM for lovastatin and 0.1 microM or 1 microM for simvastatin. DNA synthesis was decreased by more than 80% in the presence of 1 microM of lovastatin or simvastatin. In contrast, under these conditions, pravastatin had no influence on cell proliferation or DNA synthesis, which is probably related to the lack of inhibition of sterol synthesis by pravastatin on extended incubation. The three 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors did not disturb cell viability because mitochondrial dehydrogenase activity and ATP content remained proportional to the number of cells in the culture at any concentration used.

Vastatins have a distinct effect on sterol synthesis and progesterone secretion in human granulosa cells in vitro.

Lovastatin and simvastatin are strong inhibitors of cholesterol synthesis in cultured human granulosa cells, as measured within 6 days after isolation, with IC50-values of respectively 27.0 and 18.2 nM obtained after 3.5 hours of incubation with the drugs. Pravastatin is a much weaker inhibitor of cholesterol synthesis (IC50-value of 977.8 nM) in these cells. Under these conditions inhibition of cholesterol synthesis had no influence on progesterone secretion into the medium which was probably due to the presence of large cholesterol pools in the cells. To deplete these pools, granulosa cells were cultured for 7 days after which the culture medium was changed into medium supplemented with 20% lipoprotein-depleted serum to deprive the cells of exogenous cholesterol. Additionally, 30 mIU of follicle-stimulating hormone and luteinizing hormone per ml were added to stimulate the progesterone production and secretion, thereby decreasing the cholesteryl ester pools. After 48 h of incubation, culture was continued without hormones for another two days. Thereafter, the cells were preincubated for 24 h without or with 1 microM of lovastatin, simvastatin or pravastatin in medium containing lipoprotein-deficient serum and the above-mentioned hormones. This period is followed by incubation for another 24 h in the presence of [14C]acetate after which cells and media were collected for determination of 14C-labelled sterols synthesized and progesterone secreted into the media. Now, lovastatin and simvastatin, which strongly inhibited sterol synthesis, significantly attenuated the secretion of progesterone. One microM of pravastatin had no significant effect on sterol synthesis nor on progesterone secretion. When the latter experiment was performed under conditions in which exogenous cholesterol was provided in the form of human low density lipoproteins, no influence of the vastatins on progesterone secretion was observed. So under conditions in which the cholesterol pools were decreased, lovastatin and simvastatin attenuated the progesterone secretion, whereas pravastatin did not. When pools were filled by exogenous cholesterol, no effect on progesterone secretion by either of the drugs was observed.