Prevalence of clozapine-induced sialorrhea and its effect on quality of life.

RATIONALE: Clozapine has proven to be superior to other antipsychotic drugs in the treatment of schizophrenia but is under-prescribed due to its potentially severe side effects. Clozapine-induced sialorrhea (CIS) is a frequent and extremely uncomfortable side effect, which remains understudied. OBJECTIVES: To examine the prevalence of diurnal and nocturnal CIS in a sample of patients treated with clozapine, and to evaluate its impact on quality of life. METHODS: We conducted a cross-sectional, observational study of 130 patients with schizophrenia spectrum disorders treated with clozapine. The prevalence of CIS was evaluated via specific sialorrhea scales. None of the patients included in the study was receiving a specific treatment for hypersalivation during the study period. Possible associations between sialorrhea and clinical and quality of life variables were analyzed. RESULTS: Of 130 subjects, 120 (92.3%) suffered from CIS. Eighty-one (62.31%) suffered from diurnal CIS, 115 (88.56%) from nocturnal CIS, and 85 (65.38%) suffered from both. Significant positive associations between quality of life and diurnal CIS (B = 0.417; p = 2.1e - 6, R2 = 0.156) and nocturnal CIS (B = 0.411; p = 7.7e - 6, R2 = 0.139) were detected. Thirty per cent of the subjects reported a moderate to severe negative impact of sialorrhea on their quality of life. CONCLUSIONS: The present study suggests that CIS is highly prevalent in patients with schizophrenia and has an important impact on quality of life in one-third of our sample. Therefore, the inclusion of a systematic evaluation and treatment of CIS in standard clinical practice is highly recommended. TRIAL REGISTRATION: Clinical Trials ( https://clinicaltrials.gov ) under reference NCT04197037.

Molecular evidence for the nuclear localization of FADD.

The Fas-associated death domain (FADD) adaptor protein FADD/Mort-1 is recruited by several members of the tumor necrosis factor receptor (TNFR) superfamily during cell death activated via death receptors. Since most studies have focused on the interaction of FADD with plasma membrane proteins, FADD's subcellular location is thought to be confined to the cytoplasm. In this report, we show for the first time that FADD is present in both the cytoplasm and the nucleus of cells, and that its nuclear localization relies on strong nuclear localization and nuclear export signals (NLS and NES, respectively) that reside in the death-effector domain (DED) of the protein. Specifically, we found that a conserved basic KRK35 sequence of the human protein is necessary for FADD's nuclear localization, since disruption of this motif leads to the confinement of FADD in the cytoplasm. Furthermore, we show that the leucine-rich motif LTELKFLCL28 in the DED is necessary for FADD's nuclear export. Functionally, mutation of the NES of FADD and its seclusion in the nucleus reduces the cell death-inducing efficacy of FADD reconstituted in FADD-deficient T cells.

Protein kinase C regulates FADD recruitment and death-inducing signaling complex formation in Fas/CD95-induced apoptosis.

Activation of protein kinase C (PKC) triggers cellular signals that inhibit Fas/CD95-induced cell death in Jurkat T-cells by poorly defined mechanisms. Previously, we have shown that one effect of PKC on Fas/CD95-dependent cell death occurs through inhibition of cell shrinkage and K(+) efflux (Gómez-Angelats, M., Bortner, C. D., and Cidlowski, J. A. (2000) J. Biol. Chem. 275, 19609-19619). Here we report that PKC alters Fas/CD95 signaling from the plasma membrane to the activation of caspases by exerting a profound action on survival/cell death decisions. Specific activation of PKC with 12-O-tetradecanoylphorbol-13-acetate or bryostatin-1 induced translocation of PKC from the cytosol to the membrane and effectively inhibited cell shrinkage and cell death triggered by anti-Fas antibody in Jurkat cells. In contrast, inhibition of classical PKC isotypes with Gö6976 exacerbated the effect of Fas activation on both apoptotic volume decrease and cell death. PKC activation/inhibition did not affect anti-Fas antibody binding to the cell surface, intracellular levels of FADD (Fas-associated protein with death domain), or c-FLIP (cellular FLICE-like inhibitory protein) expression. However, processing/activation of both caspase-8 and caspase-3 and BID cleavage were markedly blocked upon PKC activation and, conversely, were augmented during PKC inhibition, suggesting a role for PKC upstream of caspase-8 processing and activation. Analysis of death-inducing signaling complex (DISC) formation was carried out to examine the influence of PKC on recruitment of both FADD and procaspase-8 to the Fas receptor. PKC activation blocked FADD recruitment and caspase-8 activation and thus DISC formation in both type I and II cells. In contrast, inhibition of classical PKCs promoted the opposite effect on the Fas pathway by rapidly increasing FADD recruitment, caspase-8 activation, and DISC formation. Together, these data show that PKC finely modulates Fas/CD95 signaling by altering the efficiency of DISC formation.

Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis.

The movement of intracellular monovalent cations has previously been shown to play a critical role in events leading to the characteristics associated with apoptosis. A loss of intracellular potassium and sodium occurs during apoptotic cell shrinkage establishing an intracellular environment favorable for nuclease activity and caspase activation. We have now investigated the potential movement of monovalent ions in Jurkat cells that occur prior to cell shrinkage following the induction of apoptosis. A rapid increase in intracellular sodium occurs early after apoptotic stimuli suggesting that the normal negative plasma membrane potential may change during cell death. We report here that diverse apoptotic stimuli caused a rapid cellular depolarization of Jurkat T-cells that occurs prior to and after cell shrinkage. In addition to the early increase in intracellular Na(+), (86)Rb(+) studies reveal a rapid inhibition of K(+) uptake in response to anti-Fas. These effects on Na(+) and K(+) ions were accounted for by the inactivation of the Na(+)/K(+)-ATPase protein and its activity. Furthermore, ouabain, a cardiac glycoside inhibitor of the Na(+)/K(+)-ATPase, potentiated anti-Fas-induced apoptosis. Finally, activation of an anti-apoptotic signal, i.e. protein kinase C, prevented both cellular depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the inability of cells to repolarize via inhibition of the Na(+)/K(+)-ATPase is a likely regulatory component of the death process.

Cell volume regulation in immune cell apoptosis.

The loss of cell volume is an early and fundamental feature of programmed cell death or apoptosis; however, the mechanisms responsible for cell shrinkage during apoptosis are poorly understood. The loss of cell volume is not a passive component of the apoptotic process, and a number of experimental findings from different laboratories highlight the importance of this process as an early and necessary regulatory event in the signaling of the death cascade. Additionally, the loss of intracellular ions, particularly potassium, has been shown to play a primary role in cell shrinkage, caspase activation, and nuclease activity during apoptosis. Thus, an understanding of the role that ion channels and plasma membrane transporters play in cellular signaling during apoptosis may have important physiological implications for immune cells, especially lymphocyte function. Furthermore, this knowledge may also have an impact on the design of therapeutic strategies for a variety of diseases of the immune system in which apoptosis plays a central role, such as oncogenic processes or immune system disorders. The present review summarizes our appreciation of the mechanisms underlying the early loss of cell volume during apoptosis and their association with downstream events in lymphocyte apoptosis.

Selective loss of nucleoside carrier expression in rat hepatocarcinomas.

Evidence that hepatoma cell lines show differential expression of concentrative nucleoside transporters (CNT1 and CNT2) prompted us to study the transporter proteins in 2 models of hepatocarcinogenesis, the chemically induced Solt and Farber model and the albumin-SV40 large T antigen (Alb-SV40) transgenic rat. CNT1 expression was lower in tumor biopsy specimens from Alb-SV40 rat livers than in normal tissue. Immunocytochemistry revealed that the CNT1 protein was indeed absent in the tumor lesions. CNT1 was also absent in a cell line, L25, derived from the Alb-SV40 transgenic rat liver tumors, whereas another cell line, L37, derived from the normal-appearing parenchyma, retained the expression of both carrier isoforms. The protein expression correlated with the nucleoside transport properties of these cell lines. Moreover, although CNT2 expression was highly dependent on the growth characteristics of the 2 cell lines, as was CNT1 (albeit to a lower extent) in L37 cells, it was not expressed in L25 cells at any stage of cell growth. In contrast to the transgenic model of hepatocarcinogenesis, in the chemically induced tumors the expression of CNT2 was lower, although still detectable. In summary, these data indicate that hepatocarcinogenesis leads to a selective loss or diminished expression of nucleoside carrier isoforms, a feature that may be relevant to our understanding of the molecular basis of the bioavailability of those drugs that are nucleoside derivatives and may be substrates of these carriers. The transport properties and isoform-expression profile of the L25 and L37 cell lines make them suitable hepatocyte culture models with which to study nucleoside transport processes and drug sensitivity.

Protein kinase C (PKC) inhibits fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. A role for PKC upstream of caspases.

Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with Gö6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and Gö6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with Gö6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.

Mutational analysis of three tumor suppressor genes in two models of rat hepatocarcinogenesis.

An albumin-simian virus 40 (SV40) large T-antigen (T-Ag) transgenic model and a chemically induced model of multistage hepatocarcinogenesis were created in our laboratory to study the molecular mechanisms involved in the genesis and progression of neoplasia in the rat liver. In the study presented here, these two models of rat hepatocarcinogenesis were used to perform a comparative mutational analysis of three tumor suppressor genes involved in hepatic neoplastic growth. By using polymerase chain reaction-single strand conformation polymorphism analysis and sequencing, exons 5-8 of the p53 tumor suppressor gene and a region between nt 4325 and 4479 of the rat mannose 6-phosphate/insulin-like growth factor 2 receptor (M6p/Igf2r) coding sequence were screened. The latter is homologous to the human M6P/IGF2r coding sequence which is mutated in human hepatocellular carcinoma. A complete single strand conformation polymorphism analysis of the entire coding region of the rat adenomatous polyposis coli (Apc) gene was also performed for the first time in rat tumorigenic samples. Twenty-six chemically induced rat hepatocellular carcinomas, 21 neoplasms from the livers of SV40 T-Ag animals, and five immortalized hepatic cell lines from the transgenic rats were evaluated. None of the hepatic tumors exhibited mutations in the regions analyzed. The albumin-SV40 T-Ag transgenic cell line L-60, derived from normal hepatic tissue, had two mutations in contiguous codons of exon 5 of the p53 gene: a GGT --> GTT missense transversion in codon 183 and a silent mutation in codon 184. The transversion, which may affect the DNA binding domain of the p53 protein, probably originated during cell culture and may have been positively selected because it gave a growth advantage to the mutated cells. The studied region of the M6p/Igf2r gene was not found to be mutated in these two models of rat hepatocarcinogenesis. Although M6p/Igf2r, Apc, and p53 have been shown to be mutated in a variety of human hepatic proliferative diseases, our results indicate that aberrations in these genes may not be necessary for liver carcinogenesis in the rat.

Cytoskeletal-dependent activation of system A for neutral amino acid transport in osmotically stressed mammalian cells: a role for system A in the intracellular accumulation of osmolytes.

System A activity for neutral amino acid transport is increased after hypertonic shock in NBL-1 (an epithelial cell line) and CHO-K1 cells (a nonepithelial cell line) by a mechanism which is consistent with the synthesis of a regulatory protein that activates preexisting system A carrier proteins (Ruiz-Montasell et al., 1994, Proc. Natl. Acad. Sci. USA, 91,9569-9573). In this study, we have further investigated this biological response by determining the role of cytoskeletal structures in system A regulation by hypertonic stress. Using inhibitors of the microfilament and microtubule networks, we show that the increase in system A activity after hypertonic treatment requires the integrity of both cytoskeletal structures in NBL-1 cells, although the increase in system A activity triggered by amino acid starvation is completely insensitive to any of these drugs. In contrast, the enhancement of system A activity in osmotically stressed CHO-K1 cells is not sensitive to inhibitors of the microtubule network. In both cell types, the results suggest that the inhibitors block the increase of system A activity. System A transport decreases when CHO-K1 cells return to isotonic conditions by a mechanism that is insensitive to inhibitors of protein and mRNA synthesis. The increase in system A transport activity is also followed by the accumulation of neutral amino acids (fourfold for alanine), which is totally blocked by the same agents (cycloheximide and actinomycin D) that prevent the increase in system A activity after hypertonic treatment, thus indicating that system A is crucial for maintaining a high concentration of organic osmolytes inside the cell.

Nucleoside uptake in rat liver parenchymal cells.

Rat liver parenchymal cells express Na(+)-dependent and Na(+)- independent nucleoside transport activity. The Na(+)-dependent component shows kinetic properties and substrate specificity similar to those reported for plasma membrane vesicles [Ruiz-Montasell, Casado, Felipe and Pastor-Anglada (1992) J. Membr. Biol. 128, 227-233]. This transport activity shows apparent K(m) values for uridine in the range 8-13 microM and a Vmax of 246 pmol of uridine per 3 min per 10(5) cells. Most nucleosides, including the analogue formycin B, cis-inhibit Na(+)-dependent uridine transport, although thymidine and cytidine are poor inhibitors. Inosine and adenosine inhibit Na(+)-dependent uridine uptake in a dose-dependent manner, reaching total inhibition. Guanosine also inhibits Na(+)-dependent uridine uptake, although there is some residual transport activity (35% of the control values) that is resistant to high concentrations of guanosine but may be inhibited by low concentrations of adenosine. The transport activity that is inhibited by high concentrations of thymidine is similar to the guanosine-resistant fraction. These observations are consistent with the presence of at least two Na(+)-dependent transport systems. Na(+)-dependent uridine uptake is sensitive to N-ethylmaleimide treatment, but Na(+)-independent transport is not. Nitrobenzylthioinosine (NBTI) stimulates Na(+)-dependent uridine uptake. The NBTI effect involves a change in Vmax, it is rapid, dose-dependent, does not need preincubation and can be abolished by depleting the Na+ transmembrane electrochemical gradient. Na(+)-independent uridine transport seems to be insensitive to NBTI. Under the same experimental conditions, NBTI effectively blocks most of the Na(+)-independent uridine uptake in hepatoma cells. Thus the stimulatory effect of NBTI on the concentrative nucleoside transporter of liver parenchymal cells cannot be explained by inhibition of nucleoside efflux.

Hormonal regulation of concentrative nucleoside transport in liver parenchymal cells.

Na(+)-dependent uridine uptake is stimulated in isolated rat liver parenchymal cells by glucagon. This effect is transient, reaches maximum levels of stimulation 10 min after hormone addition, and is dose-dependent. Glucagon action can be mimicked by agents that are able to hyperpolarize the plasma membrane (e.g. monensin) and by dibutyryl cyclic AMP. The effects triggered by glucagon, monensin and dibutyryl cyclic AMP are not additive, suggesting a common mechanism of action. 8-(4-Chloro-phenylthio)adenosine 3':5'-cyclic monophosphate (PCT), a cyclic AMP analogue but also a nucleoside analogue, markedly stimulates Na(+)-dependent uridine uptake in an additive manner to that triggered by monensin, similarly to the effect described for nitrobenzylthioinosine. Considering the roles reported for nucleosides in liver metabolism, the use of PCT as a cyclic AMP analogue should be precluded. Insulin is also about to up-regulate Na(+)-dependent uridine uptake by a mechanism which involves a stable induction of this transport activity at the plasma-membrane level. This is consistent with a mechanism involving synthesis and insertion of more carriers into the plasma membrane. It is concluded that the recently characterized hepatic concentrative nucleoside transporter is under short-term hormonal regulation by glucagon, through mechanisms which involve membrane hyperpolarization, and under long-term control by insulin. This is the first report showing hormonal modulation of the hepatic concentrative nucleoside transporter.

Long-term osmotic regulation of amino acid transport systems in mammalian cells.

Mammalian cells accumulate organic osmolytes, either to adapt to permanent osmotic changes or to mediate cell volume increase in cell cycle progression. Amino acids may serve as osmolytes in a great variety of cells. System A, a transport system for neutral amino acids, is induced after hypertonic shock by a mechanism which requires protein synthesis and gene transcription. Indirect evidence supports the view that system A activity increases due to the interaction of pre-existing A carriers with putative activating proteins. The intracellular accumulation of most neutral amino acids after hypertonic shock depends, exclusively, on the increase in system A activity. Long-term activation of system A is dependent on the integrity of cytoskeletal structures, but in a different way depending on whether cells are polarized or not.

Effect of protein malnutrition on neutral amino acid transport by rat hepatocytes during development.

Hepatocytes from suckling rats whose mothers were fed a low-protein diet (9% protein) showed a lower capacity for Na(+)-dependent L-alanine uptake [due to a decrease in maximal uptake rate (Vmax) of a low-affinity component of transport] and were not able to respond to insulin or glucagon, whereas those from suckling pups whose mothers were fed the control diet (17% protein) had already developed the ability to upregulate L-alanine transport after hormone treatment. When animals from low-protein-fed mothers were weaned onto a hypoprotein diet, the overall capacity for Na(+)-dependent L-alanine uptake (apparent Vmax) and its responsiveness to pancreatic hormones were restored. Hepatocytes from these animals showed a lower response to glucocorticoid treatment. Amino acid availability was dramatically decreased in suckling and weanling rats fed a low-protein diet. These results support the hypothesis that nutrient supply is an important factor in the proper development of hepatic transport functions during the suckling-weaning transition.

Evidence for a regulatory protein involved in the increased activity of system A for neutral amino acid transport in osmotically stressed mammalian cells.

System A for neutral amino acid transport is increased by hypertonic shock in NBL-1 cells previously induced to express system A activity by amino acid starvation. The hypertonicity-mediated effect can be blocked by cycloheximide but is insensitive to tunicamycin. The activity induced may be inactivated irreversibly by the addition of system A substrates, by a rapid mechanism insensitive to cycloheximide. In CHO-K1 cells, hypertonicity increases system A activity, as has been shown in NBL-1 cells. This effect is additive to the activity produced by derepression of system A by amino acid starvation and is insensitive to tunicamycin. Furthermore, the alanine-resistant mutant CHO-K1 alar4, which bears a mutation affecting the regulatory gene R1, involved in the derepression of system A activity after amino acid starvation, is still able to respond to the hypertonic shock by increasing system A activity to a level similar to that described in hypertonicity-induced derepressed CHO-K1 (wild type) cells. These results suggest (i) that the hypertonicity-mediated increase of system A activity occurs through a mechanism other than that involved in system A derepression and (ii) that a regulatory protein coded by an osmotically sensitive gene is responsible for further activation of preexisting A carriers.