William T. Mason

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

SummaryUsing a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.

Rab3 proteins: key players in the control of exocytosis

Although some mechanistic aspects of exocytosis, such as fusion events, have been well documented by the technique of time-resolved membrane-capacitance measurement, it was only recently that new insights into the molecular mechanisms involved in the traffic of secretory vesicles were provided by the convergence of different lines of research. In this review Lledo et al. present some of the recent findings concerning small GTPases of the Rab3 subfamily which regulate hormone release, triggered by entry of Ca2+, in endocrine and neuroendocrine cells. In view of these new results, Rab proteins might be considered as candidates for inhibition or stimulation of specific steps involved in vesicle traffic.

Imaging of intracellular calcium in rat anterior pituitary cells in response to growth hormone releasing factor.

1. Changes in intracellular ionized calcium [Ca2+]i induced by human growth hormone releasing factor (hGRF) were analysed by quantitative fluorescent microscopy using a dual‐wavelength, ratiometric video imaging system and low light level charge‐coupled device (CCD) camera visualizing Fura‐2 in dispersed male rat anterior pituitary cells. 2. In cells responding to hGRF, spontaneous basal oscillations in [Ca2+]i were frequently observed, and these were usually characterized by a gradient of [Ca2+]i localized in the subplasmalemmal region of the cell. 3. Of the cells which responded to hGRF, the peptide evoked a rise in [Ca2+]i, especially in the region of the subplasmalemma. Continuous application of 10 nM‐hGRF produced several different temporal patterns of the [Ca2+]i response which were not attributable to spatial response profiles. A sustained rise in [Ca2+]i was the most common type of response to hGRF (44% of the cells examined). 4. One‐third of the cells responding to 10 nM‐hGRF showed spontaneous basal [Ca2+]i oscillations ranging from 100 to 500 nM. Mean values of basal and 10 nM‐hGRF‐induced [Ca2+]i of these cells were 81 +/‐ 11 nM (mean +/‐ S.E.M., n = 27) and 560 +/‐ 47 nM (n = 27) respectively. There was no significant correlation between basal [Ca2+]i and the hGRF‐induced [Ca2+]i increase, nor was there any consistent correlation with regard to the spatial response profile. 5. Application of 2 mM‐Co2+ abolished the hGRF‐induced rise in [Ca2+]i. Quantitative analysis of this effect, performed by comparing the mean [Ca2+]i evoked during the application of hGRF with and without Co2+, respectively, also showed significant inhibition of the hGRF‐induced rise in [Ca2+]i by the application of Co2+ (P less than 0.001). 6. The hGRF‐induced rise in [Ca2+]i was completely suppressed by replacing extracellular Na+ with impermeant molecules such as mannitol. The onset and offset of suppression was as rapid as that induced by Co2+. Quantitative analysis showed significant inhibition of the hGRF‐induced rise in [Ca2+]i by Na+ replacement (P less than 0.01). 7. Tetrodotoxin, a potent blocker of voltage‐sensitive Na+ channels (5 and 20 microM), did not affect the hGRF‐induced rise in [Ca2+]i. 8. Extracellular application of the membrane permeable dibutyryl cyclic AMP (DBcAMP) to elevate intracellular levels of cyclic AMP caused a large rise in [Ca2+]i, which was dependent on extracellular Na+ and was abolished by 2 mM‐Co2+ applied in the bath.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of three types of potassium current in cultured neurones of rat supraoptic nucleus area.

1. Whole‐cell, voltage‐clamp recordings were obtained from neurones of the supraoptic area of neonatal rats in dissociated cell culture. Recordings were made from neurones having the same morphology as those which were vasopressin or oxytocin immunoreactive. 2. Three types of voltage‐activated K+ current were identified on the basis of their kinetics, voltage sensitivities, Ca2+ dependence and pharmacology. The currents corresponded to the delayed rectifier current (IK), the A‐current (IA), and the Ca2+‐dependent current (IK(Ca] described in other neurones. 3. IK had a threshold of ‐40 mV, a sigmoidal time course of activation, and was sustained during voltage steps lasting less than 300 ms. The underlying conductance was voltage dependent reaching a maximum at +30 mV (mean maximum conductance 4.09 nS). The activation time constant was also voltage dependent declining exponentially from 4.5 ms at ‐30 mV to 1.8 ms at +50 mV. 4. IA was transient, and was activated from holding potentials negative to ‐70 mV; the maximum conductance (mean 5.9 nS) underlying the current was obtained at +10 mV. The activation and inactivation time constants were voltage dependent: the activation time constant declined exponentially between ‐40 mV (2.2 ms) and +40 mV (0.65 ms). 5. IK and IA were attenuated by the K+ channel blockers tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP). TEA blocked the conductance underlying IK but appeared to alter the kinetics of IA. In contrast, 4‐AP blocked the conductance underlying IA and, to a lesser extent, IK. 6. IK and IA were activated independently of external Ca2+ and the voltage activation of Ca2+ channels since these currents were recorded in the presence of Co2+, a Ca2+ channel blocker. 7. IK(Ca) was recorded only when Ca2+ (2 mM) was present in the external medium. From a holding potential of ‐30 mV, IK(Ca) had a threshold of ‐20 mV, was maximal at about +20 mV and declined at more positive potentials. This current was sustained during voltage steps lasting 100 ms and was abolished by addition of Co2+ (2 mM) to the medium. 8. The possible roles of the three K+ currents in regulating the characteristic firing behaviour of supraoptic neurones previously recorded in vivo and in vitro are discussed.

Acidic calcium pools in intraerythrocytic malaria parasites

Calcium uptake by permeabilized P. chabaudi malaria parasites was measured at the trophozoite stage to assess calcium accumulation by the parasite organelles. As determined with 45Ca2+, the total calcium in the parasite was found to be 11 pmoles/10(7) cells. When the K+/H+ uncoupling agent, nigericin was present, this level fell to 6.5 pmoles/10(7) cells. A similar regulatory mechanism operates in P. falciparum, since addition of nigericin to intact parasites in calcium free-medium resulted in a transient elevation of free calcium in the parasite cytosol, as judged by fluorescent imaging of single cells loaded with the calcium indicator fluo-3,AM. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) and monensin, inhibitors of H+ ATPases and K+/H+ ionophore respectively, induced calcium elevation in fluo-3, AM-labeled intact P. chabaudi parasites. We conclude that malaria parasites utilize acidic intracellular compartments to regulate their cytosolic free calcium concentration.

Patch Clamp Recordings of Single Ion Channel Activation by Gonadotrophin-Releasing Hormone in Ovine Pituitary Gonadotrophs

Gonadotrophs of the ovine pars tuberalis have been studied using the patch clamp technique for recording of single ion channel currents. We report that gonadotrophin-releasing hormone (GnRH) acts on these cells to open an inward-current cation channel which is permeable to Ca2+. When measured in cell-attached patches with 5 mM extracellular Ca2+, the GnRH-activated channel has a unit slope conductance of 8.0 +/- 2.6 pS (range 4-14 pS). The channel conductance is increased to 13.6 +/- 2.4 pS when the external medium contains 95 mM Ba2+ as the charge carrier. GnRH action appears to be mediated through an internal messenger system, since GnRH does not need to be in direct contact with these channels in order to cause their opening. This internal messenger system is unlikely to be Ca2+ itself. In addition, two other voltage-dependent outward-current channels have also been detected, both of which are permeable to K+, but differentiated in the cell-attached recording mode by widely different conductance values of 20-30 and 100-120 pS, respectively, and a reversal potential of -90 to -100 mV. The higher conductance channel is sensitive to internal Ca2+, and its probability of opening is increased in the presence of GnRH.

Electrophysiological Recordings from Gonadotrophs

The electrophysiological properties of gonadotrophs have been studied in vitro, using the ovine adenohypophyseal pars tuberalis as a naturally enriched source of this cell type. Trypsin-dispersed pars tuberalis cells maintained in primary tissue culture had a membrane potential of -72 +/- 4 mV (mean +/- SEM) and an input resistance of 314 +/- 38 M omega. Spontaneous action potentials were not observed; however, a single spike could be induced by depolarizing current injection. The hypophysiotrophic peptide gonadotrophin-releasing hormone (GnRH) increased membrane voltage fluctuations, but these fluctuations (+/- 5-10 mV) did not induce action potentials or changes in membrane potential or resistance. Power spectra obtained from analysis of this noise indicated that the fundamental event underlying GnRH action has a mean life-time of 38.4 +/- 4.5 ms. The observations that cells incubated in recording medium secreted luteinizing hormone in response to GnRH and that the GnRH-induced increase in voltage noise was inhibited by Ca2+ channel antagonists support the hypotheses (1) that gonadotrophin secretion is initiated by GnRH-induced Ca2+ channel activation and (2) that action potentials are not a prerequisite for gonadotrophin release.

Dissociated Adult Rat Subfornical Organ Neurons Maintain Membrane Properties and Angiotensin Responsiveness for up to 6 Days

We have utilised standard dissociation techniques to obtain a preparation of subfornical organ (SFO) cells that have been maintained in tissue culture for up to 1 week. Stable (> 15 min) whole cell recordings were obtained from 80 cells displaying rapid (<2 ms) voltage-dependent sodium currents (blocked by tetrodotoxin in 10 of 10 cells tested), and current evoked action potentials, which were thus classified as SFO neurons. These neurons had a resting membrane potential of-63.8 +/- 1.3 mV (mean +/- SEM), spike amplitude of 86.8 +/- 2.5 mV, and input resistance of 1.2 +/- 0.1 G omega, characteristics which did not change significantly in recordings obtained for up to 6 days after dissociation. Current clamp recording showed that of 65 cells tested with bath application of angiotensin (ANG; 1,000-10nM), 41 responded to this peptide with decreases in input resistance (control 1.4 +/- 0.16 G omega, after ANG 0.78 +/- 0.1 G omega, p < 0.0001), and depolarisations (mean 18.3 +/- 2.0 mV, p < 0.0001). Similar recordings were obtained from viable cells up to 6 days after initial cell dissociation. These studies provide the first description of the basic membrane properties of dissociated SFO neurons. The responsiveness of these cells to ANG supports the conclusion that their properties are similar to those in vivo. These data suggest that use of this technique will permit systematic analysis of the membrane events underlying the actions of multiple ligands on this uniquely specialised group of CNS neurons.

cAMP directly facilitates Ca‐induced exocytosis in bovine lactotrophs

We have used the whole cell patch clamp technique on single prolactin‐secreting bovine lactotrophs to measure plasma membrane capacitance (C m), an index of membrane surface area, under voltage‐clamp during cytosol dialysis with Ca and cAMP. cAMP increased the magnitude and rate of Ca‐induced exocytosis (C m increase) without affecting membrane conductance; however, cAMP had no detectable effect on C m when intracellular Ca was low. We thus report new evidence that cAMP can facilitate Ca‐induced secretion in a synergistic fashion, by acting directly on the secretory apparatus, independently of membrane conductance activation.

Extracellular ATP activates calcium entry and mobilization via P2U-purinoceptors in rat lactotrophs

Extracellular ATP has been previously shown to activate calcium signalling in pituitary cell populations [1] but the particular cell types involved have not been identified. We used video imaging of Fura-2 loaded into single rat pituitary cells and identified as lactotrophs to study the effects of extracellular ATP on [Ca2+]i. ATP does not permeabilize the cells as shown by exclusion of propidium iodide. ATP causes two types of calcium transients in lactotrophs. The most common response is a rapid increase in [Ca2+]i that decays slowly and is terminated by washout of ATP. This type of response is also seen in calcium-free medium, demonstrating mobilization of calcium stores dependent upon the presence of the agonist. ATP also stimulates calcium entry as detected by Mn(2+)-quenching of Fura-2. ATP in Mg(2+)-free medium and ATP gamma S are effective agonists suggesting ATP4- is the active form. The presence of P2-purinoceptors is apparent because ATP, ADP and AMP increase [Ca2+]i in decreasing order of potency and adenosine has no effect. ATP-induced calcium transients are reduced by the P2-purinoceptor antagonists suramin and quinidine. UTP is equipotent with ATP and defines the receptor subtype as P2U. We conclude that ATP4- acts on rat lactotrophs via P2U-purinoceptors to elevate [Ca2+]i from intracellular and extracellular sources.