Global pharmacogenomics: Impact of population diversity on the distribution of polymorphisms in the CYP2C cluster among Brazilians.

The impact of biogeographical ancestry, self-reported 'race/color' and geographical origin on the frequency distribution of 10 CYP2C functional polymorphisms (CYP2C8*2, *3, *4, CYP2C9*2, *3, *5, *11, CYP2C19*2, *3 and *17) and their haplotypes was assessed in a representative cohort of the Brazilian population (n=1034). TaqMan assays were used for allele discrimination at each CYP2C locus investigated. Individual proportions of European, African and Amerindian biogeographical ancestry were estimated using a panel of insertion-deletion polymorphisms. Multinomial log-linear models were applied to infer the statistical association between the CYP2C alleles and haplotypes (response variables), and biogeographical ancestry, self-reported Color and geographical origin (explanatory variables). The results showed that CYP2C19*3, CYP2C9*5 and CYP2C9*11 were rare alleles (<1%), the frequency of other variants ranged from 3.4% (CYP2C8*4) to 17.3% (CYP2C19*17). Two distinct haplotype blocks were identified: block 1 consists of three single nucleotide polymorphisms (SNPs) (CYP2C19*17, CYP2C19*2 and CYP2C9*2) and block 2 of six SNPs (CYP2C9*11, CYP2C9*3, CYP2C9*5, CYP2C8*2, CYP2C8*4 and CYP2C8*3). Diplotype analysis generated 41 haplotypes, of which eight had frequencies greater than 1% and together accounted for 96.4% of the overall genetic diversity. The distribution of CYP2C8 and CYP2C9 (but not CYP2C19) alleles, and of CYP2C haplotypes was significantly associated with self-reported Color and with the individual proportions of European and African genetic ancestry, irrespective of Color self-identification. The individual odds of having alleles CYP2C8*2, CYP2C8*3, CYP2C9*2 and CYP2C9*3, and haplotypes including these alleles, varied continuously as the proportion of European ancestry increased. Collectively, these data strongly suggest that the intrinsic heterogeneity of the Brazilian population must be acknowledged in the design and interpretation of pharmacogenomic studies of the CYP2C cluster in order to avoid spurious conclusions based on improper matching of study cohorts. This conclusion extends to other polymorphic pharmacogenes among Brazilians, and most likely to other admixed populations of the Americas.

Telomere shortening in patients with drug-resistant epilepsy.

Epilepsy affects about 1 % of the world population. Mesial temporal lobe epilepsy (mTLE) presents with seizures initiated in hippocampus and is the most frequent form of epilepsy. About 30 % of individuals with mTLE do not respond to conventional medications maintaining seizures and consequently new lesions on a daily basis. Treatment-resistant epilepsy has a huge social and individual burden due to impaired quality of life and increased mortality rate. There are many reasons for telomere shortening in individuals with mTLE, such as a chronic mitochondrial oxidative stress and increased levels of pro-inflammatory mediators. In the past 10 years, there was a boom of studies establishing association between telomere length and chronic/complex disorders. Telomeres are essential for the maintenance of genomic integrity. Telomere length has been assumed as a biological marker for stress and cellular ageing. Here we hypothesized that individuals affected with treatment-resistant mTLE would course with a shorter telomere than controls. So, we measured leucocytes telomere length in a sample of 89 individuals, 48 treatment-resistant mTLE compared to 41 healthy controls. As expected, we observed a significant shorter telomere in the peripheral cell leukocytes of treatment-resitant mTLE group. Telomere length was not associated with sex, side of hippocampal sclerosis, family history, etiology of seizures, duration of disease or the Engel score. Our results points towards the need of further investigation to shed light on the relation of telomeres shortening and the outcomes and impacts of epilepsy.

The leukocytes expressing DARPP-32 are reduced in patients with schizophrenia and bipolar disorder.

Bipolar disorder (BPD) and schizophrenia (SCZ) are severe disorders representing an enormous social, familiar and individual burden, being SCZ the most disabling psychiatric disorder characterized by psychosis and cognitive impairment. It is well known that SCZ and BPD are associated with abnormalities in dopamine signaling pathway. Recent data in the literature have demonstrated altered expression levels of some proteins involved in the modulation of this pathway in both brain and peripheral tissues. It was shown that protein and mRNA levels of dopamine and cAMP regulated phosphoprotein (DARPP-32) were downregulated in dorsolateral prefrontal cortex (DLPFC) of patients with SCZ or BPD when compared to controls. Due to the difficulty to access brain tissue and the absence of objective laboratory tests for bio-markers, we measured DARPP-32 expression in blood cell sub-populations (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) taking advantage of the close relation of nervous and immune systems. Using flow cytometry as the analytical method, our results have shown that the DARPP-32 expression was diminished in CD4+ T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and was also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. These results showed that DARPP-32 expression in immune cells agrees with reports of reduced DARPP-32 protein in the DLPFC of BPD or SCZ patients. Our data suggest that DARPP-32 expression in PBMC could be used as a source of bio-markers to help in the treatment response of neuropsychiatry disorders as a window to the changes in the brain of those patients.

Expression of neuronal calcium sensor-1 (NCS-1) is decreased in leukocytes of schizophrenia and bipolar disorder patients.

Schizophrenia (SCZ) and bipolar disorder (BPD) are severe illnesses representing an enormous social, familiar and individual burden that affect 1% of the population world-wide. Several evidences indicate abnormalities of the dopamine system in both SCZ and BPD. Neuronal calcium sensor-1 (NCS-1) is a protein that has many functions in neurotransmission such as inhibition of dopamine D(2) receptor desensitization, regulation of ionic channels and enhancement of exocytosis of neurotransmitters. In addition, NCS-1 protein expression and mRNA levels were found increased in pre-frontal cortex (PFC) of SCZ and BPD patients. NCS-1 expression in neural and neuroendocrine cells is well documented and, recently, it was shown that NCS-1 is also expressed in mast cells and neutrophils. NCS-1 has important functions in mast cells since it stimulates Fc epsilon RI-triggered exocytosis and the release of arachidonic acid metabolites. Then, due to the known close relation between the nervous and immune systems, we sought to investigate the NCS-1 expression in lymphocytes and monocytes (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) of SCZ and BPD patients. Using flow cytometry, our results have shown that NCS-1 expression was diminished in CD4+T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. Results suggest that immune cells might be a cellular model for studies with SCZ and BPD patients considering NCS-1 functions. Efforts need to be done to investigate the motive of the decreased percentage of immune cells expressing NCS-1 in patients with SCZ and BPD.

Modulation of muscarinic signaling in PC12 cells overexpressing neuronal Ca2+ sensor-1 protein.

It has been suggested that overexpression of neuronal Ca2+ sensor-1 (NCS-1) protein is implicated in the pathophysiology of neurodisorders such as schizophrenia, bipolar disturbance and X-linked mental retardation. The mechanism by which NCS-1 would be involved in the causes and/or consequences of these neurodisorders is still far from elucidation. Independent evidence has pointed NCS-1 as a key regulator of synaptic efficacy by altering the expression and activity of voltage-gated channels, inhibiting internalization of dopaminergic receptors, and altering phosphoinositide metabolism. In this study, we examined the possible participation of NCS-1 protein in signal transmission dependent on muscarinic receptor activation, using PC12 cells stably expressing NCS-1 (PC12-NCS-1). Carbachol (CCH; 300 microM) was able to evoke glutamate release more efficiently from PC12-NCS-1 (15.3+/-1.0nmol/mg of protein) than wild type cells (PC12-wt; 8.3+/-0.9nmol/mg of protein). This increase of glutamate release induced by CCH was independent on extracellular Ca2+ influx. Additionally, a larger increase of cytoplasmic levels of InsP3 (663.0+/-63.0 and 310.0+/-39.0% of fluorescence in A.U.) and [Ca2+]i (766.4+/-40.0 and 687.8+/-37.1nmol/L) was observed after CCH stimulus of PC12-NCS-1 compared with PC12-wt. Clearly distinction between intracellular Ca2+ dynamics was also observed in PC12-NCS-1 and PC12-wt. A larger increase followed by fast decay of [Ca2+]i was observed in PC12-NCS-1. A plateau with a delayed decay of [Ca2+]i was characteristic of PC12-wt [Ca2+]i response. Both enhancement of InsP3 production and glutamate release observed in PC12-NCS-1 were blocked by atropine (10 microM). Together, our data show that overexpression of NCS-1 in PC12 cells induces an enhancement of intracellular second messenger and transmitter release dependent on CCH response, suggesting that muscarinic signaling is "up-regulated" in this cell model.

Is the 5-HTTLPR polymorphism associated with bipolar disorder or with suicidal behavior of bipolar disorder patients?

The serotonin transporter gene has a 44 bp insertion/deletion polymorphism within the promoter region (5-HTTLPR) with two allelic forms, the long (L) and the short (S) variants. Association between the low-activity S variant and bipolar disorder (BPD) has been shown but its replication has not been consistent. It has also been described as an association between the S allele and suicidal behavior. Since suicidal behavior is a rather frequent event in BPD, an important question is whether suicidality, instead of bipolarity itself, could be related to S allele. We assessed 351 subjects (167 bipolar inpatients and 184 healthy controls). Diagnosis was conducted by a psychiatrist using a structured interview (MINI-PLUS), according to DSM-IV criteria. Suicidal behavior was assessed using a semi-structured instrument and a review of medical records. Genotyping of the 5-HTTLPR was performed using PCR. There were 77 patients with a history of previous suicide attempts. Bipolar patients and healthy controls showed comparable genotypic and allelic frequencies. Patients carrying the S allele made violent suicide attempts more frequently (chi(2) = 20.2; P = 0.0001) and made more suicide attempts (t = 2.6; P = 0.01). We were able to show an association between the S allele and suicidal behavior but not with BPD. Our data suggest that a phenotypic stratification, taking into account the suicidal behavior history, is of pivotal importance when performing association studies between BPD and 5-HTTLPR genotypes, which could explain previous contradictory results.

Effects of antipsychotics on intestinal motility in zebrafish larvae.

BACKGROUND: Antipsychotics are essential for the treatment of schizophrenia. However, due to side effects, both continuity of treatment and patients' general health can be jeopardized. Some of these drugs, especially clozapine, have a class of side effects attributed to their antimuscarinic properties, such as dysmotility, a condition in which muscles of the digestive system become impaired. Dysmotility may also alter the speed, strength or coordination of the digestive organs, causing distention, disturbing gastrointestinal transit, leading to symptoms such as bloating, nausea, vomiting, and even malnutrition. In this study, our aim was to develop an in vivo assay capable of identifying and studying the antimuscarinic effects of antipsychotics in a zebrafish model. METHODS: We performed video recordings of in vivo 5-day postfertilization (dpf) zebrafish larvae gastrointestinal tracts and analyzed the frequency of spontaneous and regular cycles of contractions of the gut. KEY RESULTS: The assay was first validated with treatment with atropine. We showed that this antimuscarinic drug reduces peristaltic cycles. Subsequently, the larvae were treated with the antipsychotics haloperidol, risperidone, and clozapine. Neither haloperidol nor risperidone reduced gut motility, but clozapine significantly reduced the frequency of cycles of contractions (P<.0001), which confirms the existing clinical data. CONCLUSIONS & INFERENCES: We conclude that this zebrafish assay efficiently identifies anticholinergic side effects of antipsychotics, and can thus be a quick and useful way to screen for this property in new drugs.

Investigation of A218C tryptophan hydroxylase polymorphism: association with familial suicide behavior and proband's suicide attempt characteristics.

According to WHO, suicide accounts for about 1,000,000 deaths worldwide every year. In view of these dramatic data, several studies have tried to identify possible biological mechanisms and markers of suicide. Genes encoding for proteins involved in the serotonergic transmission are major candidates in association studies of suicidal behavior. The gene that codes for tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin, is one of these candidates. Two polymorphisms in intron 7 of this gene (A218C and A779C) have been described, but their role in suicidal behavior remains uncertain. TPH A218C polymorphism was analyzed in a sample of 248 psychiatric patients and 63 healthy controls. In addition, at least one close relative member was interviewed to assess family suicidal behavior history. Our research confirmed that a positive history of suicide attempts in a family member is associated with the chance of an individual to attempt suicide. Furthermore, we demonstrated that familial suicide attempts are more lethal and frequently more violent. We were not able to find significant differences of the TPH genotype frequencies between patients and controls. The TPH A218C genotypes were not associated with a history of suicide attempt and the lethality of the most lethal lifetime suicide attempt and suicide attempt method. The authors conclude that the A218C polymorphism of the TPH gene may not be a susceptibility factor for suicidal behavior in this group of psychiatric patients but confirm that a family suicidal behavior history increases the proband's suicide attempt risk.

GABA-induced calcium signaling in cultured enteric neurons is reinforced by activation of cholinergic pathways.

UNLABELLED: GABA is an important inhibitory transmitter in the CNS. In the enteric nervous system, however, both excitatory and inhibitory actions have been reported. Here, we investigated the effects of GABA on the intracellular Ca2+ concentration of guinea-pig myenteric neurons (at 35 degrees C) using Fura-2-AM. Neurons were identified by 75 mM K+ depolarization (5 s), which evoked a transient intracellular Ca2+ concentration increase. GABA (10 s) induced a dose dependent (5 nM-1 microM) transient intracellular Ca2+ concentration rise in the majority of neurons (500 nM GABA: 251+/-17 nM, n=232/289). Interestingly, the response to 5 microM GABA (n=18) lasted several minutes and did not fully recover. GABA response amplitudes were significantly (P<0.001) reduced by GABAA and GABAB receptor antagonists (10 microM) bicuculline and phaclofen. The GABAA agonist isoguvacine (10 microM) and GABAB agonist baclofen (10 microM) induced similar responses as 50 nM GABA, while the GABAC agonist cis-4-aminocrotonic acid (CACA) (10 microM) only elicited small responses in a minority of neurons. Removal of extracellular Ca2+ abolished all responses while depletion of intracellular Ca2+ stores by thapsigargin (5 microM) did not alter the responses to 500 nM GABA (n=13), but reduction of Ca2+ influx through voltage-dependent Ca2+ channels did. The nicotinic antagonist hexamethonium (100 microM) also reduced GABA responses by almost 70% suggesting that GABA stimulates cholinergic pathways, while the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) and the 5-HT3 receptor blocker ondansetron only had minor effects. CONCLUSION: GABA elicits transient intracellular Ca2+ concentration responses in the majority of myenteric neurons through activation of GABAA and GABAB receptors and much of the response can be attributed to facilitation of ACh release. Thus GABA may act mainly as a modulator that sets the state of excitability of the enteric nerve network. A concentration of 5 microM GABA, although frequently used in pharmacological experiments, seems to cause a detrimental response reminiscent of the neurotoxic effects glutamate has in the CNS.

Novel vasopressin type 2 (AVPR2) gene mutations in Brazilian nephrogenic diabetes insipidus patients.

Nephrogenic diabetes insipidus (NDI) is an inherited disorder characterized by renal resistance to the antidiuretic effect of arginine vasopressin (AVP), resulting in polyuria, polydipsia, and hypoosmolar urine. In the vast majority of cases, NDI is associated with germ-line mutations in the vasopressin receptor type 2 gene (AVPR2) and in about 8% of the cases with the water channel aquaporin-2 gene (AQP-2) mutations. To date, approximately 277 families with 185 germ-line mutations in the AVPR2 gene have been described worldwide. In the present study, the AVPR2 gene was genotyped in eight unrelated Brazilian kindred with NDI. In five of these NDI families, novel mutations were noted (S54R, I130L, S187R, 219delT, and R230P), whereas three seemingly unrelated probands were found to harbor previously described AVPR2 gene mutations (R106C, R137H, R337X). Additionally a novel polymorphism (V281V) was detected. In conclusion, although NDI is a rare disease, the findings of mutations scattered over the entire coding region of the AVPR2 gene are a valuable model to determine structure function relationship in G-protein-coupled receptor related diseases. Furthermore, our data indicate that in Brazil the spectrum of AVPR2 gene mutations is "family specific".

Postpartum depression symptoms among Amazonian and Northeast Brazilian women.

BACKGROUND: Studies analyzing the prevalence of postpartum depression in Brazil have recently increased. However, few studies have examined the Northeast region of Brazil, and no studies have investigated the Amazon region. Therefore, the aim of this study was to investigate postpartum depression in these two regions. METHODS: We administered the Edinburgh Postnatal Depression Scale to a total of 3060 women who used the Brazilian public health system and had given birth between one and three months prior to the interview. A cut-off score ≥11 was used to indicate symptoms of postpartum depression. After calculating the prevalence, univariate logistic regressions were performed separately for several possible risk factors (p<0.05). RESULTS: The overall rate of reported symptoms of postpartum depression was 19.5%. The prevalence in the northeast region and Amazon region were 19.0% and 20.3%, respectively (p=0.36). In the univariate logistic regression, low education level (<7 years: p<0.001; 8-10 years: p=0.003), ethnicity (Black: p=0.02; Pardo: p=0.02), few prenatal visits (1 or 2 visits: p=0.04), prenatal care self-assessed as "not very good" (p<0.001) and the prenatal care adequacy index of partially suitable (p=0.01) or not suitable (p<0.001) were identified as significant risk factors for postpartum depression symptoms. LIMITATIONS: Mothers who did not bring their children for immunization. The cross-sectional study does not allow for causality to be established. CONCLUSION: the prevalence rates of postpartum depression were similar to the rates observed for developing countries and higher than the rates observed in developed countries. Based on these findings, we recommend that screening and treatment of pregnant women should be considered a public health priority.

Alpha- and beta-scorpion toxins evoke glutamate release from rat cortical synaptosomes with different effects on [Na+]i and [Ca2+]i.

Scorpion toxins have long been used as tools in the investigation of neurotransmitter release mechanisms. We have used rat cortical synaptosomes to study the effects of a beta-type scorpion toxin (TiTX-gamma) on the release of glutamate and on the concentrations of free sodium and calcium ions inside the synaptosomes. The effects are compared with those of an alpha-type scorpion toxin (TsTX), on which there have been more studies. TsTX increased overall internal sodium and calcium ion concentrations and glutamate release in an incremental, dose dependent manner. TiTX-gamma similarly evoked glutamate release in an incremental, dose dependent manner. However, TiTX-gamma caused little increase in the overall internal sodium and calcium ion concentrations at low doses that evoked a significant release of glutamate and a maximal increase in these ions at somewhat higher doses. The results suggest that TiTX-gamma preferentially binds sodium channels close to the active zones for glutamate release and indicates that modifications of the activation or inactivation of the Na+-channel can lead to very different changes in the cytosolic concentrations of free Na+and Ca2+, with consequences for neurotransmission. This provides an interesting perspective concerning modulation of neurotransmitter release via pharmacological manipulation of Na+-channel properties, that may lead to a better comprehension of its physiological and pathological roles.

Calcium channels coupled to depolarization-evoked glutamate release in the myenteric plexus of guinea-pig ileum.

Glutamate is the major excitatory neurotransmitter in the CNS. The recent characterization of glutamate as a neurotransmitter in the enteric nervous system opened a new line of investigation concerning the role of glutamate in that system. The present study aimed to further characterize the enteric glutamate release and the calcium channels coupled to it. For this study the myenteric plexus-longitudinal muscle of guinea-pig ileum was stimulated with potassium chloride or with electrical pulses. The released glutamate was detected by spectrofluorimetry. Laser scanning confocal microscopy was used for analysis of immunolabeled enteric tissue for co-localization studies of calcium channels (N- and P/Q-type) and glutamate transporters (EAAC1). Here we report the effects of known Ca(2+)-channel blockers on glutamate release evoked by KCl-depolarization or electrical stimulation in the myenteric plexus. We find that N-type Ca(2+) channels control a major portion of evoked glutamate release from this system, with a very small contribution from L-type Ca(2+) channels. Moreover, alpha(1A)-like (P-type Ca(2+) channel) and alpha(1B)-like (N-type Ca(2+ )channel) immunoreactivity co-localized with glutamate transporters in the myenteric plexus. In addition, KCl-evoked or electrically stimulated glutamate release was sensitive to omega-agatoxin IVA, in a frequency-dependent manner, suggesting that P-type channels are also coupled to the release of glutamate. We, thus, conclude that both N-type and P-type Ca(2+) channels control most of the evoked glutamate release from the enteric nervous system, as also occurs in some parts of the CNS.

Investigation of the modulation of glutamate release by sodium channels using neurotoxins.

The modulation of neurotransmitter release by calcium channels is well established, yet, sodium channels were regarded mainly as charge carriers. Many lines of evidence suggest a more fine-tuning role played by sodium channels. Using rat cerebrocortical isolated nerve endings (synaptosomes) and two toxins that have separate sites of action on sodium channels and provoke distinct changes in channel kinetics, we were able to show that depending on the rate of increase in channel conductance, the outcome in terms of neurotransmitter release and calcium channel types coupled to that event are different. Mainly, our study focused on veratridine, an alkaloid from lilaceous plants that binds to sodium channel toxin site 2, and tityustoxin, a toxin purified from the venom of the Brazilian yellow scorpion Tityus serrulatus that binds to site 3. Veratridine induces a slower increase in intrasynaptosomal sodium and calcium concentrations, slower depolarization, delayed exocytosis and a slower and predominantly calcium-independent glutamate release, when compared to tityustoxin.Thus, we have used these two toxins to investigate the events that start with sodium entry and culminate with the release of glutamate in isolated nerve endings (synaptosomes) from rat cerebral cortex. With that in mind we measured intrasynaptosomal free sodium concentration [Na(+)](i), intrasynaptosomal free calcium concentration [Ca(2+)](i), membrane potential, exocytosis and glutamate release using fluorescent probes.

Inhibition of protein kinases prevents lymphocyte activation by Schistosoma mansoni antigens and reduces in vitro [correction of in vivo] granuloma reaction.

T-cell activation is regulated by signal transduction events initiated by protein kinases. The role of different protein kinases during stimulation of peripheral blood mononuclear cells (PBMC) from chronic intestinal schistosomiasis patients was evaluated using specific inhibitors of protein kinases. We have assayed their ability to interfere with cell proliferation, in vitro granuloma reaction and calcium mobilization. Taken together, our results suggest that Schistosoma mansoni antigen activation of PBMC involves protein kinases. The results observed could be important in the understanding of the mechanism involved in the immunomodulation of schistosomiasis.

Different effects of reducing agents on omega-conotoxin GVIA inhibition of [3H]-acetylcholine release from rat cortical slices and guinea-pig myenteric plexus.

1. The effect of reducing reagents on omega-conotoxin GVIA (omega-CgTX) inhibition of the release of [3H]-acetylcholine ([3H]-ACh) induced by tityustoxin, K+ 50 mM and electrical stimulation was investigated in rat brain cortical slices. 2. In cortical slices the inhibition of tityustoxin or electrically-stimulated [3H]-ACh release by omega-CgTX was dramatically increased by reducing reagents ascorbate or beta-mercaptoethanol. Dehydroascorbic acid did not substitute for ascorbate. 3. Depolarization induced by K+ 50 mM caused [3H]-ACh release from cortical slices which was not inhibited by omega-CgTX, even in the presence of ascorbate. 4. In the guinea-pig myenteric plexus, omega-CgTX inhibition of the tityustoxin induced release of [3H]-ACh was independent of ascorbate. 5. It is suggested that N-type-like calcium channels in guinea-pigs myenteric plexus may have pharmacological/biochemical diversity from similar channels of rat cerebral cortex.

A toxin from the spider Phoneutria nigriventer that blocks calcium channels coupled to exocytosis.

1. The aim of the present experiments was to investigate the pharmacological action of a toxin from the spider Phoneutria nigriventer, Tx3-3, on the function of calcium channels that control exocytosis of synaptic vesicles. 2. Tx3-3, in confirmation of previous work, diminished the intracellular calcium increase induced by membrane depolarization with KCl (25 mM) in rat cerebrocortical synaptosomes. The toxin was very potent (IC50 0.9 nM) at inhibiting calcium channels that regulate calcium entry in synaptosomes. In addition, Tx3-3 blocked the exocytosis of synaptic vesicles, as measured with the fluorescent dye FM1-43. 3. Using omega-toxins that interact selectively with distinct neuronal calcium channels, we investigated whether the target of Tx3-3 overlaps with known channels that mediate exocytosis. The results indicate that the main population of voltage-sensitive calcium channels altered by Tx3-3 can also be inhibited by omega-agatoxin IVA, an antagonist of P/Q calcium channels. Omega-conotoxin GVIA, which inhibits N type calcium channels did not decrease significantly the entry of calcium or exocytosis of synaptic vesicles in depolarized synaptosomes. 4. It is concluded that Tx3-3 potently inhibits omega-agatoxin IVA-sensitive calcium channels, which are involved in controlling exocytosis in rat brain cortical synaptosomes.

Inhibition of K+ channel activity by 4-AP stimulates N-type Ca2+ channels in CHP-100 cells.

The potassium channel blocker, 4-aminopyridine (4-AP), stimulates neurotransmitter release via plasma membrane depolarization and subsequent activation of voltage-gated calcium channels. The present study assessed the effects of 4-AP on intracellular calcium levels in the human neuroblastoma cell line CHP-100. Blockade of K+ channels with 4-AP significantly increased intracellular calcium concentration ([Ca2+]i). This increase occurred via activation of plasma membrane Ca2+ channels. The 4-AP induced rise in [Ca2+]i was not inhibited by the L-type Ca2+ channel blocker nifedipine but was sensitive to the N-type Ca2+ channel blocker omega-contotoxin GVIA. Tetrodotoxin did not alter the effect of 4-AP. These results suggest that in CHP-100 cells, following inhibition of K+ channels by 4-AP, N-type Ca2+ channels are activated.

Effects of sodium on PKC translocation; relationship to neurotransmitter release.

Our previous work using Na+ channel activators such tityustoxin (TsTX), indicated that local increases in Na+ modulate glutamate release from synaptosomes. We have now investigated the role of the Ca2+/phospholipid-dependent protein kinase (PKC) in mediating this effect. TsTX and KCl stimulate 'fast' glutamate release to the same extent but TsTX is more effective than KCl in enhancing the 'slow' phase of release. KCl greatly stimulates PKC translocation. However, TsTX inhibits basal and phorbol ester-induced translocation while the Na(+)-ionophore, gramicidin D, has no effect. Taken together, these data suggest TsTX mediated localized Na+ entry inhibits PKC translocation and that this effect may be associated with recruitment of vesicles to the readily releasable pool.