Pronociceptive effects induced by cutaneous application of a transient receptor potential ankyrin 1 (TRPA1) channel agonist methylglyoxal in diabetic animals: comparison with tunicamycin-induced endoplastic reticulum stress.

Methylglyoxal (MG) is a reactive carbonyl compound generated in diabetes mellitus. MG is an established transient receptor potential ankyrin 1 (TRPA1) channel agonist that contributes to TRPA1-mediated diabetic pain hypersensitivity. Here we studied whether exposure to diabetes and thereby to elevated endogenous MG modulates hypersensitivity induced by intradermal MG. Moreover, since diabetes induces endoplasmic reticulum (ER) stress, we compared the role of TRPA1 in diabetes and ER stress by assessing whether tunicamycin-induced ER stress, without diabetes, produces TRPA1-mediated pain hypersensitivity and by assessing whether ER stress and diabetes have similar modulatory effects on MG-induced hypersensitivity. In vitro patch clamp recording was performed to assess whether tunicamycin is a TRPA1 agonist. Behavioral tests showed that mechanical hypersensitivity induced by MG is reduced in diabetes and ER stress. In healthy controls, hypersensitivity induced by MG was reduced when MG was administered for the second time in the same but not adjacent plantar sites. Hypersensitivity induced by ER stress was reversed by pharmacological blocking of TRPA1. In vitro patch clamp recording indicated that tunicamycin itself (30 µM) is not a TRPA1 agonist. The results indicate that pain hypersensitivity induced by non-diabetic ER stress as well as that induced by diabetes is mediated TRPA1. Reduction of MG-induced hypersensitivity in diabetes or ER stress may, at least partly, be explained by peripheral mechanisms.

Brain-derived neurotrophic factor increases the motility of a particular N-methyl-D-aspartate /GABA-responsive subset of neural progenitor cells.

Neurotrophins like brain-derived neurotrophic factor (BDNF) promote the migration of subsets of neural progenitor cells. The mechanism by which motility is increased and the functional properties of BDNF-responsive cells are not very well known. We have used the neurosphere model, combining time-lapse microscopy, immunocytochemistry, and Ca(2+) imaging, to study the effect of BDNF on parameters such as motility and neurotransmitter responsiveness of migrating neural progenitors. At the initiation of differentiation thick glial glutamate-aspartate transporter (GLAST)-positive radial processes emerged from the neurosphere, followed by the exit of neuron-like cells. The neuron-like cells moved outside the radial processes in a phasic manner with intermittent surges of motility and stationary periods. BDNF increased the number and promoted the progress of the neuron-like cells by prolonging surges and decreasing the length of stationary phases. The average rate of cellular movement during surges was unaffected by BDNF. BDNF also caused a several fold increase in positive staining for tropomyosin-related kinase B (TrkB) receptors and neuronal markers such as Calbindin, microtubule-associated protein-2 (MAP-2), and neuron-specific nuclear protein (NeuN) in cells outside the radial network. Calcium imaging allowed for further characterization of the BDNF-responsive cell population. Kainate-responsive cells, denoting the expression of AMPA/kainate receptors, dominated in the outer migration layers while cells responding to (S)-3,5-dihydroxyphenylglycine (DHPG) via metabotropic glutamate receptor 5 (mGluR5) dominated in the inner migration layers. BDNF did not appreciably affect the distribution of these cells but promoted the redistribution of a small subpopulation (about 20%) of N-methyl-D-aspartate (NMDA)- and GABA-responsive cells to the outermost layers of migration. The results demonstrate that BDNF does not affect cell motility per se but alters the phasic behavior of cell movement by promoting periods of high motility in a defined subpopulation of cells which give a robust Ca(2+) response to NMDA and GABA.

Reduction of BDNF expression in Fmr1 knockout mice worsens cognitive deficits but improves hyperactivity and sensorimotor deficits.

Fragile X syndrome (FXS) is a common cause of inherited intellectual disability and a well-characterized form of autism spectrum disorder. As brain-derived neurotrophic factor (BDNF) is implicated in the pathophysiology of FXS we examined the effects of reduced BDNF expression on the behavioral phenotype of an animal model of FXS, Fmr1 knockout (KO) mice, crossed with mice carrying a deletion of one copy of the Bdnf gene (Bdnf(+/-)). Fmr1 KO mice showed age-dependent alterations in hippocampal BDNF expression that declined after the age of 4 months compared to wild-type controls. Mild deficits in water maze learning in Bdnf(+/-) and Fmr1 KO mice were exaggerated and contextual fear learning significantly impaired in double transgenics. Reduced BDNF expression did not alter basal nociceptive responses or central hypersensitivity in Fmr1 KO mice. Paradoxically, the locomotor hyperactivity and deficits in sensorimotor learning and startle responses characteristic of Fmr1 KO mice were ameliorated by reducing BNDF, suggesting changes in simultaneously and in parallel working hippocampus-dependent and striatum-dependent systems. Furthermore, the obesity normally seen in Bdnf(+/-) mice was eliminated by the absence of fragile X mental retardation protein 1 (FMRP). Reduced BDNF decreased the survival of newborn cells in the ventral part of the hippocampus both in the presence and absence of FMRP. Since a short neurite phenotype characteristic of newborn cells lacking FMRP was not found in cells derived from double mutant mice, changes in neuronal maturation likely contributed to the behavioral phenotype. Our results show that the absence of FMRP modifies the diverse effects of BDNF on the FXS phenotype.

Suitability of Smopex-102 cation-exchange fiber for analytical purposes and drug monitoring.

The present study aimed to evaluate the suitability of Smopex-102 cation-exchange fiber for the separation of acidic and basic model drugs from biological fluids (e.g. serum) prior to chromatographic analysis. In addition, the interactions of the drugs with the fiber were studied. The study found that basic antidepressant model drugs bound to a considerably greater extent than acidic drugs to poly(acrylic acid) (PAA) grafted Smopex-102 cation-exchange fiber from 25 mM HEPES buffer (pH 7.0) and spiked serum. Drug binding from serum decreased except for acidic drugs due to drug distribution between serum proteins and cation-exchange fiber. Electrostatic interactions were possibly the most important factors affecting drug binding to the fiber. Basic drugs were released most effectively from the fiber by using acetic acid (mean released amount 123.7 +/- 36.3% and mean absolute recovery 95.4 +/- 23.8%). Results demonstrated that the cation-exchange fiber evaluated might be a potential material for separating basic drugs from protein-free and proteinaceous (e.g. serum) liquid solutions for subsequent monitoring and evaluation. However, the drug release solution and release time must be optimized more precisely in order to validate described sample preparation method for each basic drug.

Apelin, orexin-A and leptin plasma levels in morbid obesity and effect of gastric banding.

UNLABELLED: Maintenance of human energy homeostasis is regulated by a complex network. Peptides secreted from the gastrointestinal tract (GI) are signaling to the brain and other organs initiating or terminating food intake and energy expenditure. In the present study we investigated basal plasma levels of apelin, orexin-A, and leptin in morbid obese patients. In addition, we measured in a subgroup of these patients in the same individual orexin-A and leptin plasma levels one year after gastric banding surgery. METHODS: Basal plasma values were determined in obese patients (BMI=48+/-1 kg/m2n=32) after an overnight fast and compared to healthy, normal weighted (BMI=22+/-2 kg/m2n=12) controls. In addition, blood samples were collected in a subgroup of patients (BMI=48+/-1 kg/m2n=8) the day before surgery and 1 year after the operation. Apelin, orexin-A, and leptin levels were analysed using ELISAs. RESULTS: One year after the operation obese patients significantly lost weight (from 48+/-2 kg/m2 to 39+/-2 kg/m2; p<0,001). Apelin, orexin-A and leptin levels in obese patients were significantly higher compared to control individuals (736+/-50 pg/ml vs. 174+/-14 pg/ml, p<0.0001; 75.3+/-24.1 pg/ml vs. 0.8+/-0.4 pg/ml, p<0.0001; 79.0+/-2.4 ng/ml vs. 5.8+/-0.8 ng/ml, p<0.0001, respectively). Apelin and leptin plasma concentrations also correlated significantly with BMI (r=0.769, p<0.0001; r=0.778; p<0.0001, respectively), while orexin-A correlation was rather weak (r=0.335, p<0.03). No difference between pre- and post-operative orexin-A levels was observed, while leptin plasma levels significantly decreased from 45.1+/-5.4 ng/ml to 27.3+/-6.0 ng/ml (p=0.015). CONCLUSIONS: Apelin, orexin-A, and leptin plasma levels correlated positively with the BMI. One year after gastric banding with significant loss in BMI basal plasma levels of leptin decreased, while orexin-A remained unchanged.

The STC-1 cells express functional orexin-A receptors coupled to CCK release.

Orexins are newly discovered neuropeptides regulating feeding and vigilance and have been detected in neuroendocrine cells of the gut. Potential neuroendocrine functions of orexin are unknown. Therefore, the effects of orexin-A on the intestinal neuroendocrine cell line, STC-1, were investigated as a model system. RT-PCR demonstrated the presence of both OX(1) and OX(2) receptors. Stimulation with orexin-A produced a dose-dependent release of cholecystokinin (CCK), which was abolished by removal of extracellular Ca(2+) or the presence of the voltage-gated L-type Ca(2+)-channel blocker diltiazem (10 microM). Orexin-A (Ox-A) elevated intracellular Ca(2+), which was dependent on extracellular Ca(2+). Furthermore, orexin-A caused a membrane depolarization in the STC-1 cells. Ox-A neither elevated cAMP levels nor stimulated phosphoinositide turnover in these cells. These data demonstrate a functional orexin receptor in the STC-1 cell line. Ox-A produces CCK release in these cells, by a mechanism involving membrane depolarization and subsequently activation of L-type voltage-gated Ca(2+)-channels.

Modelling of promiscuous receptor-Gi/Gs-protein coupling and effector response.

A single G-protein-coupled receptor might activate multiple G-protein species. This multiplex coupling ability can be used by tissues to regulate signalling; for the pharmacologist, such multiplex coupling might cause difficulties in the interpretation of experimental data. In this article, we present mathematical models for the activation of two separate G-protein species by a single receptor. Issues addressed concern mutual antagonism between the G proteins and the availability of an already activated receptor for interaction with a new G protein (receptor-G-protein-effector complexing versus free diffusion of G proteins) in addition to receptor-G-protein precoupling at different G-protein and receptor expression levels. The output from the receptor models uses, as readout, a new model for adenylyl cyclase regulation by two allosteric regulators (i.e. G(s) and G(i)).

Role of G-protein availability in differential signaling by alpha 2-adrenoceptors.

The impact of G-protein expression on the coupling specificity of the human alpha(2B)-adrenergic receptor (alpha(2B)-AR) was studied in Sf9 cells. The alpha(2B)-AR was shown to activate both coexpressed G(s)- and G(i)-proteins in a [(35)S]GTPgammaS binding assay. Noradrenaline and the synthetic agonist UK14,304 were equally potent and efficacious in stimulating G(i) activation. At the effector level (adenylyl cyclase), both ligands stimulated cAMP production. In the presence of forskolin, the effects of the agonists were more complex. Noradrenaline stimulated cAMP production, while UK14,304 showed a biphasic concentration-response curve with inhibition of stimulated cAMP production at low agonist concentrations and further stimulation at high agonist concentrations. G(s) coexpression caused a monophasic stimulatory response with both ligands. Coexpression with G(i) resulted in a biphasic concentration-response curve for noradrenaline and a monophasic inhibition with UK14,304. Experiments with a panel of agonists demonstrated that the more efficacious an agonist is in stimulating cAMP production, the weaker is its ability to couple to inhibition of cAMP accumulation via exogenous G(i). To be able to explain the mechanistic consequences of dual G-protein coupling described above, we developed a mathematical model based on the hypothesis that an agonist induces different conformations of the receptor having different affinity for different G-proteins. The model reproduced the profiles seen in the concentration-response curves with G(s) and G(i) coexpression. The model predicts that the affinity of the receptor conformation for G-proteins as well as the availability of G-proteins will determine the ultimate response of the receptor.

Pharmacological characterization of STKR, an insect G protein-coupled receptor for tachykinin-like peptides.

STKR is a G protein-coupled receptor that was cloned from the stable fly, Stomoxys calcitrans. Multiple sequence comparisons show that the amino acid sequence of this insect receptor displays several features that are typical for tachykinin (or neurokinin, NK) receptors. Insect tachykinin-related peptides, also referred to as "insectatachykinins," produce dose-dependent calcium responses in Drosophila melanogaster Schneider 2 cells, which are stably transfected with this receptor (S2-STKR). These responses do not depend on the presence of extracellular Ca(2+)-ions. A rapid agonist-induced increase of inositol 1,4,5-trisphosphate (IP(3)) is observed. This indicates that the agonist-induced cytosolic Ca(2+)-rise is caused by a release of Ca(2+) ions from intracellular calcium stores. The pharmacology of STKR is analyzed by studying the effects of the most important antagonists for mammalian NK-receptors on STKR-expressing insect cells. The results show that spantide II, a potent substance P antagonist, is a real antagonist of insectatachykinins on STKR. We have also tested the activity of a variety of natural insectatachykinin analogs by microscopic image analysis of calcium responses in S2-STKR cells. At a concentration of 1 microM, almost all natural analogs produce a significant calcium rise in stable S2-STKR cells. Interestingly, Stc-TK, an insectatachykinin that was recently discovered in the stable fly (S. calcitrans), also proved to be an STKR-agonist. Stc-TK, a potential physiological ligand for STKR, contains an Ala-residue (or A) instead of a highly conserved Gly-residue (or G). Arch.

Orexin receptors couple to Ca2+ channels different from store-operated Ca2+ channels.

We have investigated Ca2+ release and receptor- and store-operated Ca2+ influxes in Chinese hamster ovary-K1 cells expressing human OX1 orexin receptor. Receptor-operated Ca2+ influx-response to 3 nM orexin-A was not affected by Gd3+ or 2-APB (2-aminoethoxydiphenyl borate), but was inhibited by Ni2+. Store-operated Ca2+ influx was blocked by Ni2+, Gd3+ and 2-APB, whereas the thapsigargin-induced release was unaffected. 2-APB did not block inositol-1,4,5- trisphosphate-dependent Ca2+ release in these cells. Thus, low concentrations of orexin-A cause activation of two Ca2+ influxes in the cells: primarily, a receptor-operated Ca2+ influx, and secondarily, a store-depletion activated Ca2+ influx, which is subsequent to receptor-activated Ca2+ influx and the therewith-caused IP3 production. The results show that these two rely on different molecular entities.

2-aminoethoxydiphenyl borate reveals heterogeneity in receptor-activated Ca(2+) discharge and store-operated Ca(2+) influx.

We have investigated Ca(2+) release and receptor- and store-operated Ca(2+) influxes in Chinese hamster ovary-K1 (CHO) cells, SH-SY5Y human neuroblastoma cells and RBL-1 rat basophilic leukemia cells using Fura-2 and patch-clamp measurements. Ca(2+) release and subsequent Ni(2+)-sensitive, store-operated influx were induced by thapsigargin and stimulation of G protein-coupled receptors. The alleged noncompetitive IP3 receptor inhibitor,2-aminoethoxydiphenyl borate (2-APB) rapidly blocked a major part of the secondary influx response in CHO cells in a reversible manner. It also reduced Mn(2+) influx in response to thapsigargin. Inhibition of Ca(2+) release was also seen but this was less complete, slower in onset, less reversible, and required higher concentration of 2-APB. In RBL-1 cells, I(CRAC) activity was rapidly blocked by extracellular 2-APB whereas intracellular 2-APB was less effective. Store-operated Ca(2+) influxes were only partially blocked by 2-APB. In SH-SY5Y cells, Ca(2+) influxes were insensitive to 2-APB. Ca(2+) release in RBL-1 cells was partially sensitive but in SH-SY5Y cells the release was totally resistant to 2-APB. The results suggest, that 2-APB (1) may inhibit distinct subtypes of IP3 receptors with different sensitivity, and (2) that independently of this, it also inhibits some store-operated Ca(2+) channels via a direct, extracellular action.

High specificity of human orexin receptors for orexins over neuropeptide Y and other neuropeptides.

Orexin- and neuropeptide Y-ergic systems show physiological interaction in the regulation of appetite. In this study we investigate the postulated effect of neuropeptide Y (NPY) and other peptides directly on orexin OX1 and OX2 receptors. None of the tested peptides (NPY-variants, secretin, alpha-melanocortin, pancreatic polypeptide or pituitary adenylyl cyclase-activating peptide-38) induced any Ca2+ elevation at the concentrations up to 300 nM (human NPY, secretin and alpha-melanocortin up to 1 microM). Orexin-A- and -B-mediated Ca2+ elevations were completely unaffected by the peptides. In binding assays, human NPY, secretin and alpha-melanocortin at 1 microM did not induce any displacement of 0.1 nM [125I]orexin-A. Thus, in contrast to the previously reported result on orexin-A binding, our results demonstrate that NPY does not directly interact with orexin receptor in intact cellular settings.

Reconstitution of neurotransmission by determining communication between differentiated PC12 pheochromocytoma and HEL 92.1.7 erythroleukemia cells.

Neurotransmitter release was monitored using fura-2-loaded HEL 92.1.7 cells dispersed among differentiated PC12 cells (loaded with another Ca2+ indicator fluo-3) and immobilised using transparent polycarbonate membrane filters with uniform pore size. Depolarisation with K+ caused a rapid rise in Ca2+ concentration in the PC12 cells, followed by a delayed secondary Ca2+ response in simultaneously monitored nearby HEL cells. There was a lag period of about 20 s between the responses of the two cell types. Voltage-gated Ca2+ channels in PC12 cells were inhibited by the P/Q-type (omega-conotoxin MVIIC, omega-agatoxin IVA), N-type (omega-conotoxin GVIA) and L-type channel blockers (nifedipine) as determined using fura-2 or whole-cell patch-clamp recordings. The communication between the cell types on the other hand was sensitive to P/Q- and N-type but not to L-type channel blockers. This suggests that, as in neurons, P/Q- and N-type Ca2+ channels mediate the release of neurotransmitters acting on HEL cells. Theoretically, the procedure employed should be sensitive enough to detect single exocytotic events. Our results demonstrate that a random distribution between effector and target cells is sufficient to allow communication between cells in a manner similar to extrasynaptic transmission.

Agonist trafficking of G(i/o)-mediated alpha(2A)-adrenoceptor responses in HEL 92.1.7 cells.

1. The ability of 19 agonists to elevate Ca(2+) and inhibit forskolin-induced cyclic AMP elevation through alpha(2A)-adrenoceptors in HEL 92.1.7 cells was investigated. Ligands of catecholamine-like- (five), imidazoline- (nine) and non-catecholamine-non-imidazoline-type (five) were included. 2. The relative maximum responses were similar in both assays. Five ligands were full or nearly full agonists, six produced 20 - 70% of the response to a full agonist and the remaining eight gave lower responses (< 20%) so that their potencies were difficult to evaluate. 3. Marked differences in the potencies of the agonists with respect to the two measured responses were seen. The catecholamines were several times less potent in decreasing cyclic AMP than in increasing Ca(2+), whereas the other, both imidazoline and ox-/thiazoloazepine ligands, were several times more potent with respect to the former than the latter response. For instance, UK14,304 was more potent than adrenaline with respect to the cyclic AMP response but less potent than adrenaline with respect to the Ca(2+) response. 4. All the responses were sensitive to pertussis toxin-pretreatment. Also the possible role of PLA(2), beta-adrenoceptors or ligand transport or metabolism as a source of error could be excluded. The results suggest that the active receptor states produced by catecholamines and the other agonists are markedly different and therefore have different abilities to activate different signalling pathways.

Phenolamine-dependent adenylyl cyclase activation in Drosophila Schneider 2 cells.

Drosophila Schneider 2 (S2) cells are often employed as host cells for non-lytic, stable expression and functional characterization of mammalian and insect G-protein-coupled receptors (GPCRs), such as biogenic amine receptors. In order to avoid cross-reactions, it is extremely important to know which endogenous receptors are already present in the non-transfected S2 cells. Therefore, we analyzed cellular levels of cyclic AMP and Ca2+, important second messengers for intracellular signal transduction via GPCRs, in response to a variety of naturally occurring biogenic amines, such as octopamine, tyramine, serotonin, histamine, dopamine and melatonin. None of these amines (up to 0.1 mM) was able to reduce forskolin-stimulated cyclic AMP production in S2 cells. Furthermore, no agonist-induced calcium responses were observed. Nevertheless, the phenolamines octopamine (OA) and tyramine (TA) induced a dose-dependent increase of cyclic adenosine monophosphate (AMP) production in S2 cells, while serotonin, histamine, dopamine and melatonin (up to 0.1 mM) did not. The pharmacology of this response was similar to that of the octopamine-2 (OA2) receptor type. In addition, this paper provides evidence for the presence of an endogenous mRNA encoding an octopamine receptor type in these cells, which is identical or very similar to OAMB. This receptor was previously shown to be positively coupled to adenylyl cyclase.

Kinetic evidence for different mechanisms of interaction of black mamba toxins MT alpha and MT beta with muscarinic receptors.

By studying the influence of two toxins from the black mamba Dendroaspis polylepis on the kinetics of [3H]-N-methylscopolamine binding to muscarinic acetylcholine receptors from rat cerebral cortex, it was revealed that these toxins, MT alpha and MT beta, interact with the receptors via kinetically distinct mechanisms. MT beta bound to receptors in a one-step, readily reversible process with the dissociation constant K(d)=5.3 microM. The binding mechanism of MTalpha was more complex, involving at least two consecutive steps. A fast receptor-toxin complex formation (K(T)=3.8 microM) was followed by a slow process of isomerisation of this complex (k(i)=1.8 x 10(-2) s(-1), half-time 39 s). A similar two-step interaction mechanism has been established for a related toxin, MT2 from the green mamba D. angusticeps (K(T)=1.4 microM, k(i)=8.3 x 10(-4) s(-1), half-time 840 s). The slow isomerisation process delays the effect of MT alpha and MT2, but increases their apparent potency compared to toxins unable to induce the isomerisation process.

Measurement of chloride movement in neuronal preparations.

In this unit, protocols are described for biochemical and optical techniques that have been used by investigators to measure ligand-gated chloride movement in vesicular structures called synaptoneurosomes (also referred to as microsacs), in cultured neurons, and in the acute brain slice. These techniques can be applied to other ions as well. The measurement of uptake and efflux of radioisotopic chloride in synaptoneurosomes is used to study the responses of gamma-aminobutyric acid (GABA) receptors, which are coupled to chloride channels. Similar chloride flux assays for primary neuronal cultures are also presented. Alternatively, the efflux of chloride from synaptoneurosomes and primary neuronal cultures can be studied using fluorescent dyes and photometry. Finally, the measurement of chloride uptake can be studied in individual neurons in brain slices using fluorescent dyes and optical imaging by nonconfocal and confocal microscopy. Several support protocols are provided as well, outlining the preparation of synaptoneurosomes from specific brain regions, and the preparation, loading, and calibration of chloride-sensitive fluorescent dyes.

Oscillations in oxygen tension and insulin release of individual pancreatic ob/ob mouse islets.

AIMS/HYPOTHESIS: The role of beta-cell metabolism for generation of oscillatory insulin release was investigated by simultaneous measurements of oxygen tension (pO2) and insulin release from individual islets of Langerhans. METHODS: Individual islets isolated from the ob/ob-mice were perifused. Insulin in the perifusate was measured with a sensitive ELISA and PO2 with a modified Clark-type electrode inserted into the islets. RESULTS: In the presence of 3 mmol/l D-glucose, PO2 was 102 +/- 9 mmHg and oscillatory (0.26 +/- 0.04 oscillations/min). Corresponding insulin measurements showed oscillatory release with similar periodicity (0.25 +/- 0.02 oscillations/min). When the D-glucose concentration was increased to 11 mmol/l, PO2 decreased by 30% to 72 +/- 10 mmHg with maintained frequency of the oscillations. Corresponding insulin secretory rate rose from 5 +/- 2 to 131 +/- 16 pmol x g(-1) x s(-1) leaving the frequency of the insulin pulses unaffected. The magnitude of glucose-induced change in pO2 varied between islets but was positively correlated to the amount of insulin released (r2 = 0.85). When 1 mmol/l tolbutamide was added to the perifusion medium containing 11 mmol/l glucose no change in average oscillatory pO2 was observed despite a doubling in the secretory rate. When 8 mmol/l 3-oxymethyl glucose was added to perifusion medium containing 3 mmol/l D-glucose, neither pO2 nor insulin release of the islets were changed. Temporal analysis of oscillations in pO2 and insulin release revealed that maximum respiration correlated to maximum or close to maximum insulin release. CONCLUSION/INTERPRETATION: The temporal relation between oscillations in pO2 and insulin release supports a role for metabolic oscillations in the generation of pulsatile insulin release.

Suitability of collection tubes with separator gels for collecting and storing blood samples for therapeutic drug monitoring (TDM).

In this study, we present significant changes occurring in serum drug concentrations while using blood collection tubes that contain a barrier gel. This report also contains results with antidepressant drugs, which have not been studied before with human samples. The drug concentrations were measured either with high performance liquid chromatography (HPLC) or fluorescence polarization immunoassay (FPIA). The results show that gel tubes are suitable for blood collection for antiepileptic, antibiotic, asthma and cardioactive drug measurements, since only slight adsorption was seen (0-5%). However, the studied tubes are not suitable for blood collection of antidepressants nor benzodiazepines, because the adsorption can be 5-30%. The adsorption was even higher (up to 40%) when samples were stored for 24 h after centrifugation in gel tubes. When the centrifugation step was performed after storage the effect of the barrier gel was lower (only 0-13%). Antidepressant drug measurements performed from patient specimens collected in the studied gel tubes and stored for 3 h showed <10% adsorption of the studied drugs. After 24 h storage time, concentrations of all analysed drugs decreased even more: adsorbed amount of drugs were about 5-20%. The studied gel tubes are proposed to be satisfactory for blood collection for antidepressant drug measurements if separation step is performed within 3 h after blood clotting. With the spiked samples the adsorption to barrier gel was higher, so it seems that adsorption is faster when drugs are not highly bound to serum proteins.

The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C.

Ca(2+) elevations in Chinese hamster ovary cells stably expressing OX(1) receptors were measured using fluorescent Ca(2+) indicators fura-2 and fluo-3. Stimulation with orexin-A led to pronounced Ca(2+) elevations with an EC(50) around 1 nm. When the extracellular [Ca(2+)] was reduced to a submicromolar concentration, the EC(50) was increased 100-fold. Similarly, the inositol 1,4,5-trisphosphate production in the presence of 1 mm external Ca(2+) was about 2 orders of magnitude more sensitive to orexin-A stimulation than in low extracellular Ca(2+). The shift in the potency was not caused by depletion of intracellular Ca(2+) but by a requirement of extracellular Ca(2+) for production of inositol 1,4,5-trisphosphate. Fura-2 experiments with the "Mn(2+)-quench technique" indicated a direct activation of a cation influx pathway by OX(1) receptor independent of Ca(2+) release or pool depletion. Furthermore, depolarization of the cells to +60 mV, which almost nullifies the driving force for Ca(2+) entry, abolished the Ca(2+) response to low concentrations of orexin-A. The results thus suggest that OX(1) receptor activation leads to two responses, (i) a Ca(2+) influx and (ii) a direct stimulation of phospholipase C, and that these two responses converge at the level of phospholipase C where the former markedly enhances the potency of the latter.