Expression of the vitamin D metabolizing enzyme CYP24A1 at the annulus of human spermatozoa may serve as a novel marker of semen quality.

Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human spermatozoa. The VD-inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression at the sperm annulus. Here, we investigated if CYP24A1 expression serves as a marker for VD metabolism in spermatozoa, and whether CYP24A1 expression was associated with semen quality. We included 130 men (53 healthy young volunteers and 77 subfertile men) for semen analysis and immunocytochemical (ICC) detection of CYP24A1. Another 40 men (22 young, 18 subfertile) were tested for in vitro effects of 1,25(OH)(2)D(3) on intracellular calcium concentration ([Ca(2+)](i)) and sperm motility. Double ICC staining showed that CYP24A1 and VDR were either concomitantly expressed or absent in 80% of the spermatozoa from young men. The median number of CYP24A1-expressing spermatozoa was 1% in subfertile men and thus significantly (p < 0.0005) lower than 25% in spermatozoa from young men. Moreover, CYP24A1 expression correlated positively with total sperm count, -concentration, -motility and -morphology (all p < 0.004), and the percentage of CYP24A1-positive spermatozoa increased (15 vs. 41%, p < 0.0005) after percoll-gradient-centrifugation. We noticed that the presence of >3% CYP24A1-positive spermatozoa distinguished young men from subfertile men with a sensitivity of 66.0%, a specificity of 77.9% and a positive predictive value of 98.3%. Functional studies revealed that 1,25(OH)(2)D(3) increased [Ca(2+)](i) and sperm motility in young healthy men, while 1,25(OH)(2)D(3) was unable to increase motility in subfertile patients. In conclusion, we suggest that CYP24A1 expression at the annulus may serve as a novel marker of semen quality and an objective proxy for sperm function.

Identifying patients with therapy-resistant depression by using factor analysis.

INTRODUCTION: Attempts to identify the factor structure in patients with treatment-resistant depression have been very limited. METHODS: Principal component analysis was performed using the baseline datasets from 3 add-on studies [2 with repetitive transcranial magnetic stimulation and one with transcranial pulsed electromagnetic fields (T-PEMF)], in which the relative effect as percentage of improvement during the treatment period was analysed. RESULTS: We identified 2 major factors, the first of which was a general factor. The second was a dual factor consisting of a depression subscale comprising the negatively loaded items (covering the pure depression items) and a treatment resistant subscale comprising the positively loaded items (covering lassitude, concentration difficulties and sleep problems). These 2 dual subscales were used as outcome measures. Improvement on the treatment resistant subscale was 40% in the active treatment group compared to 17-30% improvement in the sham treatments. DISCUSSION: It is possible to describe patients with therapy-resistant depression by a factor structure. Both rTMS and T-PEMF had a clinical effect on the factor-derived scales when compared to sham treatment.

EDC IMPACT: Chemical UV filters can affect human sperm function in a progesterone-like manner.

Human sperm cell function must be precisely regulated to achieve natural fertilization. Progesterone released by the cumulus cells surrounding the egg induces a Ca2+ influx into human sperm cells via the CatSper Ca2+-channel and thereby controls sperm function. Multiple chemical UV filters have been shown to induce a Ca2+ influx through CatSper, thus mimicking the effect of progesterone on Ca2+ signaling. We hypothesized that these UV filters could also mimic the effect of progesterone on sperm function. We examined 29 UV filters allowed in sunscreens in the US and/or EU for their ability to affect acrosome reaction, penetration, hyperactivation and viability in human sperm cells. We found that, similar to progesterone, the UV filters 4-MBC, 3-BC, Meradimate, Octisalate, BCSA, HMS and OD-PABA induced acrosome reaction and 3-BC increased sperm penetration into a viscous medium. The capacity of the UV filters to induce acrosome reaction and increase sperm penetration was positively associated with the ability of the UV filters to induce a Ca2+ influx. None of the UV filters induced significant changes in the proportion of hyperactivated cells. In conclusion, chemical UV filters that mimic the effect of progesterone on Ca2+ signaling in human sperm cells can similarly mimic the effect of progesterone on acrosome reaction and sperm penetration. Human exposure to these chemical UV filters may impair fertility by interfering with sperm function, e.g. through induction of premature acrosome reaction. Further studies are needed to confirm the results in vivo.

Chemical UV Filters Mimic the Effect of Progesterone on Ca2+ Signaling in Human Sperm Cells.

Progesterone released by cumulus cells surrounding the egg induces a Ca2+ influx into human sperm cells via the cationic channel of sperm (CatSper) Ca2+ channel and controls multiple Ca2+-dependent responses essential for fertilization. We hypothesized that chemical UV filters may mimic the physiological action of progesterone on CatSper, thus affecting Ca2+ signaling in human sperm cells. We examined 29 UV filters allowed in sunscreens in the United States and/or the European Union for their ability to induce Ca2+ signals in human sperm by applying measurements of the intracellular free Ca2+ concentration. We found that 13 UV filters induced a significant Ca2+ signal at 10 µM. Nine UV filters induced Ca2+ signals primarily by activating the CatSper channel. The UV filters 3-benzylidene camphor (3-BC) and benzylidene camphor sulfonic acid competitively inhibited progesterone-induced Ca2+ signals. Dose-response relations for the UV filters showed that the Ca2+ signal-inducing effects began in the nanomolar-micromolar range. Single-cell Ca2+ measurements showed a Ca2+ signal-inducing effect of the most potent UV filter, 3-BC, at 10 nM. Finally, we demonstrated that the 13 UV filters acted additively in low-dose mixtures to induce Ca2+ signals. In conclusion, 13 of 29 examined UV filters (44%) induced Ca2+ signals in human sperm. Nine UV filters primarily activated CatSper and thereby mimicked the effect of progesterone. The UV filters 3-BC and benzylidene camphor sulfonic acid competitively inhibited progesterone-induced Ca2+ signals. In vivo exposure studies are needed to investigate whether UV filter exposure affects human fertility.

Membrane potential and cytosolic free calcium levels modulate acetylcholine-induced inositol phosphate production in insulin-secreting BTC3 cells.

Effects of membrane potential and cytosolic free Ca2+ concentrations ([Ca2+]i) on acetycholine (ACh)-induced inositol phosphate production were investigated in insulin secreting betaTC3 cells. ACh (10 microM) caused a rapid inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and increase in [Ca2+]i reaching a maximum within 5 s. The rise in Ins(1,4,5)P3 production was reduced by 79 +/- 5% when [Ca2+]i was kept low in cells loaded with the Ca2+ chelator BAPTA. The ACh-evoked Ins(1,4,5)P3 production also depended on the membrane potential as it was reduced by 31 +/- 6% in cells hyperpolarized by diazoxide, an opener of ATP-sensitive K+ channels. The Ca2+ ionophore ionomycin caused a rapid increase in [Ca2+]i and in the cellular Ins(1,4,5)P3 content. We conclude that stimulation-induced changes in membrane potential and [Ca2+]i play an important role in controlling Ins(1,4,5)P3 production in insulin-secreting betaTC3 cells.

Fluorescence labeling of the human erythrocyte anion transport system.

The anion transport system of human red cells was isolated in vesicles containing the original membrane lipids and the 95 000 dalton polypeptides (band 3) by the method of Wolosin et al. (J. Biol. Chem. (1977) 252, 2419--2427). The vesicles have a functional anion transprot system since they display sulfate transport that is inhibited by the fluorescent probe 8-anilinonaphthalene 1-sulfonate (ANS) with similar potency as in red cells. The vesicles were labeled with the SH-specific probe fluorescein mercuric acetate (FMA). Labeling lowers FMA fluorescence, and is prevented or reversed by dithiothreitol, suggesting that the reaction is with a thiol group on the protein. Fluorescnece titrations show a maximum labeling stoichiometry of 1.3 +/- 0.4 mol FMA/mol 95 000 dalton polypeptide. The polarization of bound FMA fluorescence is high indicating that the probe is highly immobilized. Pretreatment with Cu2+ + o-phenanthroline under conditions that crosslink band 3 in ghosts decreases FMA labeling 50%. Differences in kinetics of FMA labeling in sealed and leaky vesicles suggest that the reactive SH group is located in the intravesicular portion of the protein (corresponding to the cytoplasmic surface of the red cell) and that FMA can cross the membrane. Inhibitors of anion transport have no effect on FMA labeling kinetics suggesting it is not transported via the anion transport system. Sulfate transport in the labeled vesicles remains fully functional. We detected self-energy transfer between bound FMA molecules by fluorescence depolarization. With excitation at 450--50 nm P decreases from 0.4, when less than half of the proteins are labeled, to 0.1 at saturation. This depolarization is not observed with red edge excitation (510--530 nm). Addition of 0.1% sodium dodecyl sulfate (SDS) changes P to 0.32, regardless of the excitation wavelength or degree of saturation with FMA. These results indicate that the band 3 proteins are close enough to allow energy transfer between fluorophores(Ro = 37.4 A), which does not occur upon red edge excitation or when the proteins are separated by SDS. We conclude that the functional anion transport system exists as a dimer or higher oligomer of band 3 proteins in these membranes, confirming previous suggestions derived using other methods. Future applications are discussed.

Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting beta TC3-cells by enhancement of intracellular calcium mobilization.

In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (> or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action.

Anion-coupled Na efflux mediated by the human red blood cell Na/K pump.

The red cell Na/K pump is known to continue to extrude Na when both Na and K are removed from the external medium. Because this ouabain-sensitive flux occurs in the absence of an exchangeable cation, it is referred to as uncoupled Na efflux. This flux is also known to be inhibited by 5 mM Nao but to a lesser extent than that inhibitable by ouabain. Uncoupled Na efflux via the Na/K pump therefore can be divided into a Nao-sensitive and Nao-insensitive component. We used DIDS-treated, SO4-equilibrated human red blood cells suspended in HEPES-buffered (pHo 7.4) MgSO4 or (Tris)2SO4, in which we measured 22Na efflux, 35SO4 efflux, and changes in the membrane potential with the fluorescent dye, diS-C3 (5). A principal finding is that uncoupled Na efflux occurs electroneurally, in contrast to the pump's normal electrogenic operation when exchanging Nai for Ko. This electroneutral uncoupled efflux of Na was found to be balanced by an efflux of cellular anions. (We were unable to detect any ouabain-sensitive uptake of protons, measured in an unbuffered medium at pH 7.4 with a Radiometer pH-STAT.) The Nao-sensitive efflux of Nai was found to be 1.95 +/- 0.10 times the Nao-sensitive efflux of (SO4)i, indicating that the stoichiometry of this cotransport is two Na+ per SO4=, accounting for 60-80% of the electroneutral Na efflux. The remainder portion, that is, the ouabain-sensitive Nao-insensitive component, has been identified as PO4-coupled Na transport and is the subject of a separate paper. That uncoupled Na efflux occurs as a cotransport with anions is supported by the result, obtained with resealed ghosts, that when internal and external SO4 was substituted by the impermeant anion, tartrate i,o, the efflux of Na was inhibited 60-80%. This inhibition could be relieved by the inclusion, before DIDS treatment, of 5 mM Cli,o. Addition of 10 mM Ko to tartrate i,o ghosts, with or without Cli,o, resulted in full activation of Na/K exchange and the pump's electrogenicity. Although it can be concluded that Na efflux in the uncoupled mode occurs by means of a cotransport with cellular anions, the molecular basis for this change in the internal charge structure of the pump and its change in ion selectivity is at present unknown.

Ca2+ signalling in exocrine acinar cells: the diffusional properties of cellular inositol 1,4,5-trisphosphate and its role in the release of Ca2+.

The correlation between acetylcholine induced changes in the intracellular free, Ca2+ concentration ([Ca2+]i), and the inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) content in isolated acini from the rat parotid and lacrimal glands was investigated. Applying digital image processing on Fura-2 loaded acini, we observed that Ca2+ increases either simultaneously throughout the acinar configurations or that occasionally, the rise near the lumen can precede the rise near the basal part by 50-100 ms. Measurements on cell suspensions revealed a correlation between changes in [Ca2+]i and changes in the cellular Ins(1,4,5)P3 content, and it is concluded that in the individual cells Ins(1,4,5)P3 is released to the cytosol within the first second after stimulation. Applying a diffusion coefficient for cytoplasmic Ins(1,4,5)P3 of 2.83 x 10(-6) cm2/s (Allbritton et al., 1992, Science, 258, 1812-1815), we have calculated the concentration profile for this messenger in a sphere with a radius of 10 microns where Ins(1,4,5)P3 is released in the center following a monoexponential function with a rate constant of 4 s-1. Assuming that Ins(1,4,5)P3 concentrations of 1 or 5% of the maximum value is able to release Ca2+, we calculated that Ca2+ waves can appear at a rate of 100 or 40 microns/s. The present data are consistent with Ins(1,4,5)P3 being a cellular messenger, that by diffusion, initiates the Ca2+ release from the cellular pools within the first fraction of a second.

Cyclic GMP potentiates phenylephrine but not cyclic ADP-ribose-evoked calcium release from rat lacrimal acinar cells.

In the present study, we describe a role for cyclic GMP (cGMP) in the signalling pathway that leads from alpha-adrenergic receptor activation to intracellular Ca2+ mobilization in rat lacrimal acinar cells. The alpha-adrenergic agonist, phenylephrine, stimulates intracellular Ca2+ release which is blocked by inhibitors of guanylate cyclase and cGMP-dependent protein kinase Ia. The membrane-permeable cGMP analogues, dibutyryl-cGMP and 8-bromo-cGMP, potentiate ( approximately 5-fold) the Ca2+ response to submaximal phenylephrine stimulation. In contrast, the same cGMP analogues have no effect on cyclic ADP-ribose-evoked Ca2+ release from permeabilized lacrimal acinar cells. Collectively, these findings suggest that cGMP, via cGMP-dependent protein kinase I alpha , is required for intracellular Ca2+ release following alpha-adrenergic receptor activation in lacrimal acinar cells.

Cyclic ADP-ribose and inositol 1,4,5-triphosphate mobilizes Ca2+ from distinct intracellular pools in permeabilized lacrimal acinar cells.

In permeabilized lacrimal acinar cells, cyclic ADP-ribose (cADP-ribose) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) release Ca2+ in a dose dependent manner from distinct thapsigargin-sensitive Ca2+ pools. Ryanodine specifically blocks the Ca2+ response to cADP-ribose, whereas heparin strongly reduces the response to Ins(1,4,5)P3 application. GTP causes a rapid Ca2+ release by a ryanodine- and heparin-insensitive mechanism and potentiates Ins(1,4,5)P3 but not cADP-ribose evoked Ca2+ release. It is estimated that cADP-ribose can release 16 mumol Ca2+/l cells, whereas Ins(1,4,5)P3 can mobilize 55 mumol Ca2+/l cells. The results suggest that cADP-ribose and Ins(1,4,5)P3 release Ca2+ from distinct internal stores and that a third Ca2+ pool exists which can selectively interact with the Ins(1,4,5)P3-sensitive Ca2+ store by a GTP-mediated process.

Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK 293 cells induces cAMP-dependent activation of calcium-induced calcium release.

The actions of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) on cellular signalling were studied in human embryonal kidney 293 (HEK 293) cells stably transfected with the cloned human GLP-1 receptor. The cloned GLP-1 receptor showed a single high-affinity binding site (Kd = 0.76 nM). Binding of GLP-1(7-36)amide stimulated cAMP production in a dose-dependent manner (EC50 = 0.015 nM) and caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The latter effect reflected Ca(2+)-induced Ca2+ release and was suppressed by ryanodine. We propose that the ability of GLP-1(7-36)amide to increase [Ca2+]i results from sensitization of the ryanodine receptors by a protein kinase A dependent mechanism.

Desensitization of glucagon-like peptide 1 receptors in insulin-secreting beta TC3 cells: role of PKA-independent mechanisms.

1. The cellular processes involved in the desensitization of the glucagon-like peptide 1 receptors were investigated by measurements of the glucagon-like peptide 1(7-36)amide (GLP-1(7-36)amide)-induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in insulin-secreting beta TC3 cells. 2. In the presence of 11.2 mM glucose, stimulation with GLP-1(7-36)amide led to a small membrane depolarization (< 10 mV), induction of electrical activity and a rapid increase in [Ca2+]i. The increase in [Ca2+]i was not observed in the presence of the L-type Ca(2+)-channel antagonist nifedipine. However, nifedipine was ineffective when applied after addition of GLP-1(7-36)amide. 3. The increase in [Ca2+]i evoked by GLP-1-(7-36)amide was transient and even in the continued presence of the agonist, [Ca2+]i returned to the basal value within 4-5 min. The latter process was slowed, but not prevented, by inhibition of protein kinase C (PKC) by staurosporine and Ro31-8220. 4. Short pretreatment of the cells with the phorbol ester, 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA), an activator of PKC, reduced the GLP-1(7-36)amide-evoked increase in [Ca2+]i by 75%. This effect of PMA was fully reversed by staurosporine and Ro31-8220. 5. The ability of GLP-1(7-36)amide to increase [Ca2+]i disappeared upon pre-exposure of the cells to the hormone (desensitization). This process was maximal within 5 min of exposure to the agonist. Following removal of the agonist from the medium, the ability to respond to subsequent stimulation by GLP-1(7-36)amide recovered gradually with time; half and complete recovery requiring > 20 min and 60 min, respectively. The desensitizing action of GLP-1(7-36)amide persisted in the presence of either staurosporine or forskolin and did not require an elevation of [Ca2+]i. 6. Our data suggest that the GLP-1(7-36)amide-evoked increase in [Ca2+]i is initiated by Ca(2+)-influx though voltage-dependent and nifedipine-sensitive L-type Ca2+ channels but depends principally on Ca2+ mobilization from internal stores for its maintenance. The desensitization of the GLP-1 receptors that occurs in the continued presence of the agonist does not result from the activation of protein kinase A or Ca(2+)-dependent kinases/phosphatases. Our data indicate that activation of PKC may contribute to the desensitization of the GLP-1 receptors but that other (PKC-independent) mechanisms also participate in this process.

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells.

1. The effects of extracellular adenosine 5'-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5'-triphosphate (2MeSATP) and uridine 5'-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.

Anticholinergic effects of cis-chlorprothixene characterized in rat parotid acini.

The anticholinergic effects of the antipsychotic drug, cis-chlorprothixene, on the secretory events underlying the formation of primary saliva were investigated. The neuroleptic, cis-chlorprothixene, is used extensively as a major tranquillizer but shares side-effects such as xerostomia with most antidepressants. The inhibitory effects of cis-chlorprothixene upon the cholinergic-induced rise in Ca2+ as well as on O2 consumption and Cl- loss were investigated in isolated rat parotid acini in order to characterize its anticholinergic effects quantitatively. The cholinergic-induced rise in cytosolic, free Ca2+ was inhibited by cis-chlorprothixene with half-maximal effect at 1.9 microM and maximal inhibition at 10 microM. When the cytosolic, free Ca2+ was enhanced in the presence of 10 microM cis-chlorprothixene by means of the Ca2+ ionophore A23187, a loss of Cl- was observed similar to that observed during cholinergic stimulation in the absence of cis-chlorprothixene. The findings are consistent with the possibility that cis-chlorprothixene exerts its effects on the steps leading from agonist binding to the acetylcholine receptor and to the increase of cytosolic free Ca2+. Thus, measurement of the stimulation-induced rise in cytosolic, free Ca2+ in the presence of neuroleptics such as the thioxanthenes represents a fast and reliable method for detecting inhibitory effects on autonomic receptor activation.

Inhibitory effects of amitriptyline on the stimulation-induced Ca2+ increase in parotid acini.

The present study demonstrates the effects of the antidepressant, amitriptyline, and the acetylcholine antagonist, atropine, on the stimulation-induced rise in cytosolic, free Ca2+ (Cai2+). The changes in Cai2+ of collagenase-isolated rat parotid acini were measured by means of the Ca2(+)-sensitive dye, fura-2. It was found that stimulation by carbachol resulted in a maximal increase of 582 +/- 34 nM (mean +/- S.E.) in Cai2+ with a ks of 5.8 +/- 1.3 microM. Adrenaline caused a rise of 380 +/- 22 nM in Cai2+ with a ks of 0.5 +/- 0.2 microM. Amitriptyline and atropine were found to inhibit the carbachol-induced rise in Cai2+ with dissociation constants (kI) of 105 and 1.25 nM, respectively, in the absence of agonist. The adrenergic-induced rise in Cai2+ was inhibited by amitriptyline with a kI of 45 nM. Amitriptyline was found to inhibit both receptor classes by a competitive or mixed type of inhibition. Similarly, atropine exerted the same type of inhibition on the acetylcholine receptor. Amitriptyline and atropine were found to be mutually exclusive for competing for substrate binding on the receptor. These findings are consistent with a common binding site for amitriptyline and atropine on the acetylcholine receptor, possibly in close proximity with, but different from the substrate binding site. The stimulation-induced cell shrinkage evoked by the loss of electrolytes and water from the acini was measured by a 90 degree light scattering signal. It was found that this method makes possible the detection of autonomic side-effects of antidepressants on acini suspended in protein-containing media.

Reduction in the rate of inositol 1,4,5-trisphosphate synthesis in rat parotid acini by lithium.

Stimulation of muscarinic cholinergic receptors on rat parotid acinar cells causes a rapid production of inositol phosphates, with the key metabolic event being the breakdown of phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. Here a high-performance liquid chromatographic technique was used to measure the effects of intracellular lithium ions on the amount of various inositol phosphates produced. When acini were stimulated maximally with acetylcholine (ACh), the sum of all inositol phosphates produced followed a monoexponential function with a production rate constant for Ins(1,4,5)P3 of 0.07 +/- 0.01 solidus/sec. The presence of 23 mM LiCl intracellularly reduced the production rate constant of Ins(1,4,5)P3 induced by ACh to 0.03 +/- 0.01 solidus/sec, resulting in a decrease in the Ins(1,4,5)P3 production as well as in the magnitude of the rise in the intracellular free Ca2+ concentration. The lithium ion (Li+) did not affect the rate of conversion of Ins(1,4,5)P3 to either inositol 1,4-bisphosphate or inositol 1,3,4,5-tetrakisphosphate. The rate of the inositol phosphate production after the addition of the Ca2+ ionophore ionomycin was unaffected by intracellular Li+ (23 mM), which implies that the action of Li+ was at the muscarinic cholinergic receptor, on G-protein or on the interactions between G-proteins and phospholipase C. Thus, in the early events after receptor stimulation with ACh, Li+ causes a reduction in the concentration of the cellular messengers Ins(1,4,5)P3 and Ca2+.

ADP ribosyl cyclase activity in rat parotid acinar cells.

Cyclic ADP-ribose is an intracellular compound responsible for Ca2+ release in a wide variety of cell types. It may be implicated in releasing Ca2+ from ryanodine-sensitive pools in exocrine acinar cells. A bifunctional enzyme CD38 can synthesize cADP-ribose and we have characterized its properties by applying a technique in which nicotinamide guanine dinucleotide (NGD+) is used as a substrate for the synthesis of fluorescent cyclic GDP-ribose. This reaction mimics the physiologically relevant reaction in which nicotinamide adenine dinucleotide (NAD+) is converted into non-fluorescent cyclic ADP-ribose. Using NGD+ as a substrate, the reaction shows a half maximal rate of synthesis at 2.6 microM and is competitively inhibited by NAD+ with a k(i) of 12.6 microM. This reveals that both NGD+ and NAD+ are converted by CD38 to their cyclic nucleotides. We have used this fluorescence technique to characterize the extent to which parotid acinar cells contain enzymes capable of synthesizing this class of cyclic nucleotides. We found that after treatment of acinar cells with a detergent which releases intracellular enzymes, NGD+ is converted into its fluorescent derivative with a half maximal rate of synthesis at 16 microM. This reaction is also competitively inhibited by NAD+ with a k(i) of 10 microM. The data indicate that parotid acinar cells contain an enzyme capable of synthesizing the Ca2+ releasing compound, cyclic ADP-ribose. This finding suggests that cyclic ADP-ribose could play a role in Ca2+ release processes from internal stores--an important event in stimulus-secretion coupling.

Repetitive transcranial magnetic stimulation (rTMS) in combination with escitalopram in patients with treatment-resistant major depression: a double-blind, randomised, sham-controlled trial.

BACKGROUND: The role of high-frequency rTMS over the left cortex as an add-on strategy in the treatment of major depression is still uncertain even in patients resistant to pharmacotherapy. We had planned a large sham TMS controlled study in the acute phase with a placebo-controlled relapse-prevention phase with escitalopram. However, because a recent meta-analysis showed only a small effect size of rTMS over sham TMS in the acute treatment phase of depressed patients, we decided to make an interim analysis. METHOD: In patients with medication-resistant major depression we administered in a randomised trial 15 sessions of sham-controlled rTMS over three weeks in combination with 20 mg escitalopram daily. After the last rTMS, the patients were followed for another 9 weeks on 20 mg escitalopram daily. The antidepressant effect was measured by the HAM-D(6) as primary outcome scale. RESULTS: A total of 45 patients with complete data were randomised so that 23 patients received sham TMS and 22 patients received active, high-frequency rTMS over the left cortex. Over the 3 weeks, the active rTMS treatment was superior to sham TMS with effect sizes on the HAM-D(6) above 0.70, which indicates not only a statistically but also a clinically significant effect. The patients had typically been through two failed antidepressant treatment attempts with non-tricyclics before inclusion in the study. Both the rTMS and escitalopram were well-tolerated. CONCLUSION: High-frequency rTMS over the left cortex is an add-on strategy of clinical significance in combination with escitalopram in patients with major depression resistant to non-tricyclic antidepressants.