Droplet digital PCR of serum miR-499, miR-21 and miR-208a for the detection of functionally relevant coronary artery disease.

BACKGROUND: microRNAs (miRNAs) have shown promise as potential new biomarkers for myocardial injury and myocardial ischemia. New digital polymerase chain reaction (PCR) techniques allow for highly precise and reliable absolute direct quantification. METHODS: In this pilot study we used droplet digital PCR (ddPCR) to assess if miRNAs might be released into circulation in patients with functionally relevant coronary artery disease (CAD). Blood samples for measurement of high-sensitivity cardiac troponin I (hs-cTnI) and miRNAs were obtained before, immediately after peak stress, and 2 h after stress testing in a blinded manner in consecutive patients referred for rest/stress myocardial perfusion single-photon emission tomography/computer tomography (MPI-SPECT/CT). ddPCR was used to directly quantify the serum concentrations of miR-21, miR-208a, and miR-499 as potential markers of myocardial injury/ischemia. Functionally relevant CAD was determined by expert interpretation of MPI-SPECT/CT, coronary angiography and fractional flow reserve, if performed. RESULTS: Overall, 200 patients were included and functionally relevant CAD was detected in 85 of them (42%). Neither miR-21, miR-208a, nor miR-499 concentrations differed at rest, stress, or 2-h after stress when comparing patients with versus without functionally relevant CAD, while hs-cTnI concentrations were significantly higher in patients with functionally relevant CAD (P < 0.001). Exercise-induced changes in miRNA or hs-cTnI concentrations did not have diagnostic utility and were similar in patients with versus without functionally relevant CAD. CONCLUSION: miR-208a, miR-21 and miR-499 concentrations at rest, after exercise and exercise-induced changes do not provide additional clinical value regarding the detection of functionally relevant CAD.

Hypericum perforatum L. (St John's wort) extract Ze 117 inhibits dopamine re-uptake in rat striatal brain slices. An implication for use in smoking cessation treatment?

Classic synthetic antidepressant drugs, as well as St John's wort extract (SJW), directly inhibit the re-uptake of norepinephrine (NE) and/or serotonin (5-HT) into pre-synaptic axons. With chronic treatment they induce adaptive changes in a number of neurotransmitter receptors in synaptic membranes. The immediate effects of SJW Ze 117, an extract low in hyperforin content, on the specific dopamine (DA) uptake were studied in rat striatal brain slices and compared with the effects on NE and 5-HT uptake in rat cortical brain slices. Specific DA uptake was inhibited in a dose dependent manner. In contrast to the findings in synaptosomal preparations published so far, the extract showed different inhibitory potencies for the respective transporters. The potencies for the uptake inhibition of NA, DA and 5-HT were 30, 7 and 1, respectively. The results indicate that the SJW Ze 117 extract interferes in three ways with the individual uptakes of the relevant neurotransmitters that are considered to be causal in the development of depression. This observation, the concomitant and potent inhibition of DA re-uptake by SJW extract, may additionally provide a rationale for the treatment of nicotine or drug addiction with SJW.

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds.

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

St. John's wort extract Ze 117 (Hypericum perforatum) inhibits norepinephrine and serotonin uptake into rat brain slices and reduces 3-adrenoceptor numbers on cultured rat brain cells.

Despite almost forty years of widespread use, the mode of action of antidepressant drugs is still largely unknown. There is agreement that these drugs interact with central neurotransmission. Common findings are acute inhibitory actions on reuptake mechanisms for norepinephrine (NE) and for serotonin (5-HT) at presynaptic axons and chronic adaptive effects on neurotransmitter receptors on postsynaptic membranes. In particular, beta-adrenoceptor downregulation has been observed after chronic treatment with most antidepressants in vivo and in cell culture systems. We studied the effectiveness of Ze 117 (St. John's wort) extract (Hypericum perforatum) on NE- and 5-HT-uptake into rat brain slices. Potency and efficacy of the Ze 117 extract were compared with those of tricyclic (TCA) and selective serotonin reuptake inhibitor (SSRI)-type antidepressants. A dose-dependent inhibition was seen on NE and 5-HT uptake into brain slices. The Ze 117 extract was more selective for the uptake of NE than for that of 5-HT. The maximal extent of uptake inhibition by Ze 117 extract was comparable to that of imipramine (IMI), desipramine (DMI) or fluvoxamine for 5-HT, but lower for NE transport, than that of the synthetic antidepressants. Chronic exposure (8 days) of confluent C6-cell cultures to Ze 117 extract resulted in a dose-dependent beta-adrenoceptor downregulation equal to that induced by DMI, a potent TCA. None of these effects could be achieved with either hypericin or hyperforin alone in a relevant dose range. Our results indicate that the St. John's wort extract Ze 117 contains active, but as yet unknown antidepressant principles with effects comparable to those of TCAs.

Differential regulation of bradykinin receptor density, intracellular Ca2+, and prostanoid release in skin and foreskin fibroblasts. Effects of cell density and interleukin-1alpha.

1. Bradykinin (BK) receptors, cytosolic Ca2+, and prostanoids were studied in human skin and foreskin fibroblasts. 2. Bmax values of BK receptors were higher in foreskin than in skin fibroblasts, increasing with cell densities in both cell types. IL-1alpha-dependent receptor induction was blocked by cycloheximide. 3. BK-stimulated cytosolic Ca2+ elevation was higher in confluent than in non-confluent cultures and larger in foreskin than in skin fibroblasts. Responses were not enhanced after IL-1-alpha-induced up-regulation of BK receptors. 4. Intrinsic prostanoid production was higher in foreskin than in skin fibroblasts at comparable cell densities. In foreskin, but not in skin fibroblasts, BK stimulation increased the release of PGE2 10 fold and that of 6-oxo-PGF1alpha 6-7 fold. 5. Preincubation with IL-1alpha had a marked effect on prostanoid release in foreskin fibroblasts only. Subsequent BK stimulation increased the release of PGE2 and 6-oxo-PGF1alpha 7-10 fold in skin fibroblasts while this increase was only 30% in foreskin fibroblasts. Release of TXA2 reached values up to one third of the other prostanoids. The IL-1alpha induced rise in BK-stimulated PGE2 synthesis was fully abolished by specific inhibition of cyclo-oxygenase 2. 6. IL-1alpha sensitized BK-stimulated prostanoid synthesis and modulated prostanoid patterns differently in fibroblasts from skin and foreskin. The IL-1alpha effects on prostanoid release were not related to BK receptor numbers nor to the BK-stimulated Ca2+ signal but appear to be due to induction of prostanoid synthesizing enzymes. Foreskin fibroblasts seem to be unique and significantly different from fibroblasts of other skin locations in respect to their response to inflammation-associated kinins and cytokines.

Inhibition by vitamin E of drug accumulation and of phospholipidosis induced by desipramine and other cationic amphiphilic drugs in human cultured cells.

1. Cationic amphiphilic drugs (CADs) are widely used in chronic pharmacotherapies in spite of frequently observed side effects connected with lysosomal phospholipid (PL) storage. 2. It has recently been shown that alpha-tocopherol (alpha-Toc) inhibits drug- and PL accumulation in cell cultures chronically exposed to the CAD, amiodarone. 3. The mechanisms of alpha-Toc action on drug kinetics and PL storage were studied in human cultured fibroblasts exposed to single and repetitive doses of desipramine and other CADs. 4. alpha-Toc did not influence the initial, pH-dependent rapid phase of drug uptake. It inhibited, in a dose-dependent manner, the slow and the cumulative phases of drug uptake and coincidently the accumulation of cellular PLs. 5. The inhibitory effects of alpha-Toc on CAD and PL accumulations depends on the ratio between CAD and alpha-Toc concentrations in the medium. This points to competition between alpha-Toc and CADs for PL complex formation. 6. Effectiveness of alpha-Toc on drug uptake varies among different CADs. It depends on its structural integrity but is independent of stereoisomerism. The inhibitory action is restricted to the piggyback slow drug uptake and therefore related to the proportion of membrane-mediated transport to permeation into lysosomes (rapid uptake). This proportion differs among CADs. 7. alpha-Toc prevents lysosomal membrane-PL storage, accelerates disintegration of PL-stores and normalizes drug-related increased membrane fluidity. This strongly suggests that alpha-Toc restores membrane recycling, impaired by CAD exposure. 8. It remains to be tested in vivo whether alpha-Toc reduces CAD side effects without interfering with drug effectiveness.

The contribution of lysosomal trapping in the uptake of desipramine and chloroquine by different tissues.

Cationic amphiphilic drugs strongly accumulate in tissues of different organs. Uptake is controlled by two major mechanisms, non-specific binding to membrane phospholipids, and ion-trapping within acidic cellular compartments. The aim of this study was to assess the individual contributions of these two mechanisms on the uptake in vitro of desipramine and chloroquine into tissue slices of control and desipramine-treated rats. Drug uptake into intact slices was compared with uptake into slices with destroyed or non-functional acidic compartments. The sequence of desipramine uptake by tissue slices of eight different organs was: lungs > brain > heart > diaphragm > kidneys > skeletal muscles > adipose tissue > liver. The low desipramine concentration in liver may be due to metabolism of the parent drug by cytochrome P-450. Uptake of chloroquine differed widely between slices of different organs with the sequence: lungs > kidneys = brain = liver > diaphragm = heart = skeletal muscles > adipose tissue. Destruction or inactivation of the acidic compartments by homogenization and freeze-thawing or by ammonium chloride, sodium fluoride, or monensin, reduced drug uptake to similar extents. The reduction was organ-specific and may represent the size of the lysosomal compartment in the respective tissue cells. Uptake of chloroquine was more affected than that of desipramine, suggesting that ion-trapping is the main factor for chloroquine accumulation, while binding to membrane phospholipids, is the main factor for desipramine uptake. Single or multiple-dose treatments of rats with desipramine hardly had any effect on consecutive desipramine uptake into lung and liver slices, while the accumulation of chloroquine was enhanced in these slices. In conclusion, the extent of uptake of cationic amphiphilic drugs into tissue slices was tissue-specific, and the contribution of the two uptake mechanisms was strongly drug-dependent.

Vitamin E reduces accumulation of amiodarone and desethylamiodarone and inhibits phospholipidosis in cultured human cells.

Chronic administration of amiodarone (AMIO), widely used by clinicians for the treatment of therapy-resistant cardiac arrhythmias, is frequently associated with serious side-effects. AMIO and its main metabolite desethylamiodarone (DEA) are known to induce phospholipidosis in vivo and in cultured cells presumably by inhibition of lysosomal phospholipid degradation. D-alpha-Tocopherol = vitamin E (alpha-TOC) was able to reduce AMIO and DEA toxicity in cell cultures. Results from the present study showed that alpha-TOC reduced phospholipidosis in cultured human skin fibroblasts chronically exposed to micromolar concentrations of AMIO and DEA and inhibited cumulative uptake of the drugs in a dose-dependent manner. A linear correlation was observed between cellular AMIO levels and phospholipid accumulation suggesting a stoichiometric relationship. alpha-TOC was also effective in clearing previously accumulated phospholipids after discontinuation of the drug treatment. The results can best be explained by an interference of alpha-TOC (a) with drug-phospholipid complex formation responsible for both phospholipid storage and drug accumulation, and (b) with pre-existing drug-phospholipid complexes, accelerating their dissociation and rendering phospholipids to substrates for lysosomal phospholipases. The finding raises hope that side-effects of AMIO and DEA can be prevented or made reversible by the administration of alpha-TOC. It must, however, be proven that the antiarrhythmic drug will still be effective.

Distribution and density of alpha- and beta-adrenergic receptor binding sites in the bovine mammary gland.

Radioreceptor binding studies were designed to localize and determine the number of alpha- and beta-adrenergic receptors in the mammary gland of lactating cows. 3H-prazosin, 3H-rauwolscine and 3H-dihydroalprenolol were used for the regional characterization of alpha 1-, alpha 2- and beta-adrenergic receptors by competitive inhibition of binding of 3H-ligands with unlabelled adrenergic agonists and antagonists. The alpha 1-, alpha 2- and beta 2-adrenergic receptor subtypes could thus be demonstrated in the regions of the teats, large mammary ducts and parenchyma. Tissues of the teat wall, of the large mammary ducts above the gland cistern and of the mammary parenchyma were prepared to determine the density of alpha 1-, alpha 2- and beta-receptors by saturation binding assays using 3H-prazosin, 3H-rauwolscine and 3H-dihydroalprenolol respectively. Binding to high affinity sites was reversible within minutes and saturable. Equilibrium was reached within minutes. The number of alpha 1- and alpha 2-adrenergic receptors decreased from the teat to the mammary ducts to the parenchyma. Most of the alpha 1- and alpha 2-adrenergic receptors were found in the teat wall, whereas in the parenchyma alpha-adrenergic receptors were absent or barely detectable. The density of beta-adrenergic receptors was similar in the teat wall and the large mammary ducts, but much lower in the parenchyma. Thus, alpha 1-, alpha 2- and beta-adrenergic receptors were found mainly in the milk purging system and hardly at all in mammary parenchyma. Inhibition of milk removal by alpha-adrenergic stimulation is possibly due to constriction of teat wall and to constriction of the mammary ducts, whereas enhanced milk flow after beta-adrenergic stimulation is possibly due to relaxation not only of the teat sphincter and teat wall, but probably also of the large mammary ducts.

Evidence for lysosomotropism of memantine in cultured human cells: cellular kinetics and effects of memantine on phospholipid content and composition, membrane fluidity and beta-adrenergic transmission.

Memantine, an amantadine derivative, is therapeutically used for the treatment of various neurological and psychiatric disorders such as Parkinson's disease, spasticity, and dementia. Pharmacokinetics of memantine and its effects on phospholipid content and composition, on membrane properties and functions such as fluidity and beta-adrenergic transmission were studied in cultured human fibroblasts and macrophages. The kinetic behaviour of memantine was characteristic for a lysosomotropic drug. Fibroblasts exposed to 14C-memantine in the microM range accumulated the drug up to 200 fold above initial medium concentrations. Lysosomal drug storage was proven by indirect evidence and by analyses of subcellular fractions. Repetitive exposure to memantine resulted in a cumulative uptake. While memantine uptake after single exposure was fully reversible, the rate and extent of release of chronically accumulated drug was reduced but could be enhanced by the addition of unlabelled memantine or ammonium chloride to the medium. Chronic, but not single, exposure to memantine above 10 microM resulted in a concentration dependent phospholipid accumulation and in a shift in the phospholipid composition. There was an overproportionate increase in phosphatidylinositol at the expense of phosphatidylserine and sphingomyelin. Chronic exposure of cultured cells to memantine increased fluidity in the superficial layers of the plasma membrane and reduced the isoproterenol-stimulated cAMP-response without affecting beta-adrenoceptor density. All these findings were compatible with the kinetic behaviour and the effectiveness expected of a weak lysosomotropic drug.

Cellular accumulation of amiodarone and desethylamiodarone in cultured human cells. Consequences of drug accumulation on cellular lipid metabolism and plasma membrane properties of chronically exposed cells.

Amiodarone (AMIO), a potent antiarrhythmic drug, is clinically widely used despite its frequent side effects after chronic administration. These side effects coincide with an intralysosomal accumulation of AMIO and its main metabolite desethylamiodarone (DEA) and may be causally related to the drug-induced intracellular storage of phospholipids (PL). Kinetics of cellular uptake and release of radiolabelled AMIO and DEA were studied following single and multiple exposures of cultured human skin fibroblasts to 5 and 10 microM drug concentrations. AMIO and DEA were efficiently taken up into cultured cells. The rate of uptake was slower than that of other cationic amphiphilic drugs. The intracellular steady state concentrations were in the millimolar range suggesting a lysosomal trapping. Repetitive exposures of cultures resulted in a cumulative and partly saturable drug uptake. The accumulation of DEA was higher than that of AMIO throughout. AMIO and DEA previously taken up into the cells during a 2 hr exposure were completely released into the washing media, suggesting an exchangeable form of the accumulated drugs. Following repetitive exposures only part of the drugs was released. Under chasing conditions using washing media containing non-labelled AMIO and DEA respectively or ammonium chloride the release of the chronically accumulated 14C-labelled drugs was increased. This suggested a drug storage in the form of complexes in acidic compartments. Phospholipid (PL) content as well as individual PL fractions were changed in whole cells and in isolated plasma membranes. PL accumulation is assumed to occur by inhibition of PL degradation due to formation of non-degradable drug-PL complexes or by inhibition of phospholipase activities. Cellular PL accumulation seemed to interfere with PL recycling. Changes in PL composition of purified plasma membranes were in part complementary to the ones in whole cells. The alterations in membrane PL composition may explain the changes in membrane fluidity and the decrease in beta-adrenoceptor density and in isoproterenol-stimulated cAMP formation. The results obtained provide an explanation for the pharmacokinetic, and possibly for the pharmacodynamic and also toxicological behaviour of AMIO and DEA in vivo.

Cultured human skin fibroblasts modify their plasma membrane lipid composition and fluidity according to growth temperature suggesting homeoviscous adaptation at hypothermic (30 degrees C) but not at hyperthermic (40 degrees C) temperatures.

Mammalian cell metabolism is responding to changes in temperature. Body temperature is regulated around 37 degrees C, but temperatures of exposed skin areas may vary between 20 degrees C and 40 degrees C for extended periods of time without apparent disturbance of adequate cellular functions. Cellular membrane functions are depending from temperatures but also from their lipid environment, which is a major component of membrane fluidity. Temperature-induced changes of membrane fluidity may be counterbalanced by adaptive modification of membrane lipids. Temperature-dependent changes of whole cell- and of purified membrane lipids and possible homeoviscous adaptation of membrane fluidity have been studied in human skin fibroblasts cultured at 30 degrees C, 37 degrees C, and 40 degrees C for ten days. Membrane anisotropy was measured by polarized fluorescence spectroscopy using TMA-DPH for superficial and DPH for deeper membrane layers. Human fibroblasts were able to adapt themselves to hypothermic temperatures (30 degrees C) by modifying the fluidity of the deeper apolar regions of the plasma membranes as reported by changes of fluorescence anisotropy due to appropriate changes of their plasma membrane lipid composition. This could not be shown for the whole cells. At 40 degrees C growth temperature, adaptive changes of the membrane lipid composition, except for some changes in fatty acid compositions, were not seen. Independent from the changes of the membrane lipid composition, the fluorescence anisotropy of the more superficial membrane layers (TMA-DPH) increased in cells growing at 30 degrees C and decreased in cells growing at 40 degrees C.

Effects of culture and incubation conditions on membrane fluidity in monolayers of cultured cells measured as fluorescence anisotropy using trimethylammoniumdiphenylhexatriene (TMA-DPH).

Membrane fluidity of coverslip attached living cells was measured as fluorescence anisotropy using 5 microM trimethylammoniumdiphenylhexatriene (TMA-DPH) as fluorescent probe. Fluorescence anisotropy is inversely related to membrane fluidity. Cells were grown on glass coverslips that were inserted and directly incubated in quarz cuvettes. The coverslips were fixed with special holders at an angle of 30 degrees in respect to the incident light. Effects of incubation temperature, of cell growth and densities and of the ionic and nonionic composition of the incubation medium on membrane fluorescence anisotropy were measured. Membranes of growing cells were more fluid than those of stationary cells, while cell densities had no effect except at very low cell numbers. Calcium concentrations increasing from 0 to 8 mmol/l in the incubation medium proportionally decreased membrane fluidity. Hypotonicity of the incubation media increased membrane fluidity while hypertonicity compared to normotonicity had no effect. Differentiated human fibroblasts from different origins exhibited similar membrane fluidities. They were, however, different from those of rat cells. Membrane fluidity of rat brain tumor cells increased with age in culture while membrane fluidity of primary differentiating rat brain cells decreased in with age in culture. Measurement of fluorescence anisotropy in living cells attached to glass coverslips is a convenient tool to study effects of culture--as well as of environmental--conditions on membrane fluidity.

Single and multiple desipramine exposures of cultured cells. Changes in cellular anisotropy and in lipid composition of whole cells and of plasma membranes.

Effects of the antidepressant drug desipramine (DMI) on fluorescence anisotropy were studied in living cultured human fibroblasts, rat brain astrocytes and rat ROC-1 hybridoma cells (oligodendrocytes x C6). Fluorescence anisotropy, a measure for fluidity, was measured by means of a fluorescence polarization technique using a set of n-(9-anthroyloxy) fatty acids as markers. Apparent fluorescence anisotropies were determined in cells following single or multiple dose exposures to 5 microM DMI at 37 degrees and compared to control cells. In all three cell types single doses of DMI led to significant decreases in anisotropies of the deeper layers (12-AS) of the membranes only, suggesting increases in fluidity. Repeated exposures to 5 microM DMI led to cell specific, significant changes in anisotropies of the superficial membrane layers, as determined by 2-AP, 6-, 7- and 9-AS. The resulting anisotropy values of the three different cell types became more alike than prior to DMI exposure. Alterations in anisotropies were accompanied with changes in the phospholipid patterns of whole cells and isolated plasma membrane vesicles. The changes of PC/PE ratios were consistent with changes observed in fluorescence anisotropies. Such alterations may be individual regulatory responses of the cells to the chronic presence of the drug within the membranes.

Differences in hepatic drug accumulation and enzyme induction after chronic amiodarone feeding of two rat strains: role of the hydroxylator phenotype?

1. It has previously been shown that the extent of hepatic phospholipidosis induced by chronic amiodarone treatment correlates with the degree of drug accumulation in liver tissue. 2. To investigate a possible influence of pharmacogenetic factors, biochemical and morphological investigations were carried out in two rat strains differing in debrisoquine hydroxylation. 3. Plasma and liver tissue concentrations of amiodarone and its main metabolite, desethyl-amiodarone, were significantly higher in rats with deficient hydroxylation. Microsomal enzyme induction, drug cytochrome P-450 complex formation and typical ultrastructural features of phospholipidosis were only seen in rats with deficient hydroxylation and in a more sensitive species, the guinea-pig. 4. It remains to be seen whether deficient debrisoquine hydroxylation in man is associated with an increased susceptibility to amiodarone side effects.

Amiodarone-induced hepatic phospholipidosis: correlation of morphological and biochemical findings in an animal model.

Morphological and biochemical investigations were performed in guinea pigs after 1, 3, 5 and 16 weeks of amiodarone feeding. The most prominent morphological finding was an increase in dense bodies in hepatocytes, Kupffer cells and in bile duct epithelia, reaching a maximum after 5 weeks of treatment according to morphometric analysis. Similar time courses were observed for the serum and liver tissue concentrations of amiodarone and desethylamiodarone and the--albeit minimal--extent of hepatocellular necrosis. Phospholipids in the liver homogenate were unchanged after 1 week, but significantly increased after prolonged amiodarone treatment. There was no significant alteration in the pattern of individual phospholipids. Serum and tissue concentrations as well as the extent of phospholipidosis do not appear to be a function of the duration of drug application. A very close correlation, however, was observed between the liver tissue concentration of amiodarone and the amount of dense bodies as a morphological expression of phospholipidosis.

Organ-specific, qualitative changes in the phospholipid composition of rats after chronic administration of the antidepressant drug desipramine.

Rats were chronically treated with daily i.p. injections of 10 mg/kg desipramine for 21 days. A 30% decrease in the number of beta-adrenoceptors was observed in brain. A receptor desensitization of similar extent was noted in submaxillary glands and lung. No change in beta-adrenoceptor number was present in heart. Total phospholipid contents were not altered in these organs after chronic drug treatment. However, organ-specific changes were found in the phospholipid composition of submaxillary glands, lung and liver but not in whole brain and heart. The changes were variable but an increase in phosphatidylinositol and decreases in phosphatidylethanolamine and sphingomyelin were consistent. Possible alterations in the phospholipid composition of the brain might have been masked by the large and stable pool of myelin phospholipids. A casual relationship between changes in the phospholipid composition and beta-adrenoceptor desensitization is discussed.

Subcellular distribution of the antidepressant drug desipramine in cultured human fibroblasts after chronic administration. Drug-effect on the subcellular distribution of accumulated phospholipids.

Desipramine (DMI) is an important antidepressant drug and a lysosomotropic substance. In cultured fibroblasts it interferes with lysosomal functions, e.g. phospholipid degradation. Chronic exposure of cells with DMI induces storage of phospholipids. Subcellular fractionations of cultured human fibroblasts that had been exposed to a short pulse of 3H-DMI showed accumulation of DMI in two acidic compartments, one of high density represented the lysosomes and one of much lower density may contain pinosomes. In chronically exposed cells DMI accumulated in the subcellular fractions of lower density only. DMI induced an important shift of lysosomal enzymes from vesicles of high density to the ones of lower density. Phospholipids were accumulating in those vesicles of lower density as well as in the fractions that contained plasma membranes. DMI also accumulated in one part of the Golgi vesicles of acute and chronically exposed cells. In the latter phospholipids and arylsulfatase A activity were also accumulating. DMI possibly interferes with membrane recycling. This eventually could induce changes in phospholipid content and composition in the plasma membrane which may have important implications for membrane functions.

Chronic exposure of human cells in culture to the tricyclic antidepressant desipramine reduces the number of beta-adrenoceptors.

The effects of the antidepressant drug desipramine (DMI) on the density of beta-adrenoceptor sites were studied on intact cultured human cells: skin fibroblasts, lung fibroblasts and macrophages. Direct binding studies were performed with the radioligand 3H-CGP 12177, a hydrophilic beta-adrenergic antagonist. The confluent cell cultures were exposed to DMI and all three cell types showed a dose-dependent decrease in the number of beta-adrenergic binding sites. This receptor desensitisation was only seen after chronic exposure of the cells to DMI. The extent of desensitisation was comparable to that seen in brain following chronic treatment of rats with DMI. The affinity of the binding sites to the radioligand was not affected by the antidepressant drug action. From these results we suggest that the in vivo effect of antidepressant drugs on postsynaptic beta-adrenoceptor density, at least in part, reflects a primary drug action and not only an adaptive change to presynaptic events.

Human fibroblast cultures as a tool for the study of antidepressant drug action.

Studies and interpretations of results of antidepressant drug action in brain are hampered by the complexity of the target organ. Biochemical and physiological effects of these drugs can, however, be studied profitably in cultured cells. We have investigated the effects of the tricyclic antidepressant desipramine on cultured human fibroblasts. The desipramine-induced changes were compared with effects of further antidepressants and of drugs with other therapeutic indications. In addition to the drug uptake and release, effects on phospholipid metabolism and on beta-adrenoceptor density were studied. The results showed an accumulation of desipramine within the lysosomal compartment and an inhibition of the phospholipid degradation resulting in a specific change of the cellular phospholipid-pattern. Chronic but not single dose exposure to desipramine reduced the number of beta-adrenoceptors and the isoproterenol-stimulated cAMP-response. This beta-adrenoceptor desensitization is thought to be causally related to the specific change of the phospholipid-pattern induced by the drug. It can be speculated that such a drug specific change of the phospholipid-matrix may be responsible for the alterations of a number of neurotransmitter receptors and of other membrane-associated functions, observed in brains of chronically drug treated animals.