Microdomain Ca2+ activation during exocytosis in Paramecium cells. Superposition of local subplasmalemmal calcium store activation by local Ca2+ influx.

In Paramecium tetraurelia, polyamine-triggered exocytosis is accompanied by the activation of Ca2+-activated currents across the cell membrane (Erxleben. C., and H. Plattner. 1994. J. Cell Biol. 127:935-945). We now show by voltage clamp and extracellular recordings that the product of current x time (As) closely parallels the number of exocytotic events. We suggest that Ca2+ mobilization from subplasmalemmal storage compartments, covering almost the entire cell surface, is a key event. In fact, after local stimulation, Ca2+ imaging with high time resolution reveals rapid, transient, local signals even when extracellular Ca2+ is quenched to or below resting intracellular Ca2+ concentration ([Ca2+]e, < or = [Ca2+]i). Under these conditions, quenched-flow/freeze-fracture analysis shows that membrane fusion is only partially inhibited. Increasing [Ca2+], alone, i.e., without secretagogue, causes rapid, strong cortical increase of [Ca2+]i but no exocytosis. In various cells, the ratio of maximal vs. minimal currents registered during maximal stimulation or single exocytotic events, respectively, correlate nicely with the number of Ca stores available. Since no quantal current steps could be observed, this is again compatible with the combined occurrence of Ca2+ mobilization from stores (providing close to threshold Ca2+ levels) and Ca2+ influx from the medium (which per se does not cause exocytosis). This implies that only the combination of Ca2+ flushes, primarily from internal and secondarily from external sources, can produce a signal triggering rapid, local exocytotic responses, as requested for Paramecium defense.

Calcium in ciliated protozoa: sources, regulation, and calcium-regulated cell functions.

In ciliates, a variety of processes are regulated by Ca2+, e.g., exocytosis, endocytosis, ciliary beat, cell contraction, and nuclear migration. Differential microdomain regulation may occur by activation of specific channels in different cell regions (e.g., voltage-dependent Ca2+ channels in cilia), by local, nonpropagated activation of subplasmalemmal Ca stores (alveolar sacs), by different sensitivity thresholds, and eventually by interplay with additional second messengers (cilia). During stimulus-secretion coupling, Ca2+ as the only known second messenger operates at approximately 5 microM, whereby mobilization from alveolar sacs is superimposed by "store-operated Ca2+ influx" (SOC), to drive exocytotic and endocytotic membrane fusion. (Content discharge requires binding of extracellular Ca2+ to some secretory proteins.) Ca2+ homeostasis is reestablished by binding to cytosolic Ca2+-binding proteins (e.g., calmodulin), by sequestration into mitochondria (perhaps by Ca2+ uniporter) and into endoplasmic reticulum and alveolar sacs (with a SERCA-type pump), and by extrusion via a plasmalemmal Ca2+ pump and a Na+/Ca2+ exchanger. Comparison of free vs total concentration, [Ca2+] vs [Ca], during activation, using time-resolved fluorochrome analysis and X-ray microanalysis, respectively, reveals that altogether activation requires a calcium flux that is orders of magnitude larger than that expected from the [Ca2+] actually required for local activation.

Subplasmalemmal Ca2+ stores of probable relevance for exocytosis in Paramecium. Alveolar sacs share some but not all characteristics with sarcoplasmic reticulum.

Isolated subplasmalemmal Ca2+ stores ('alveolar sacs') from Paramecium tetraurelia cells sequester 45Ca2+ depending on ATP concentration. 45Ca2+ uptake is sensitive to SERCA-type Ca(2+)-ATPase inhibitors. They cause a slow release of 45Ca2+, as does caffeine. Of some importance are also the negative results we obtained with ryanodine, inositol 1,4,5-trisphosphate (InsP3), cyclic adenosinediphosphoribose (cADPR), 3',5'-cyclic guanosine monophosphate (cGMP, +/- beta-nicotinamide-adenine dinucleotide) or with increased [Ca2+]. These data were corroborated by experiments in vivo, including microinjection studies. Again ryanodine, InsP3, cADPR or cGMP did not trigger exocytosis, the trigger effect of SERCA inhibitors was sluggish, whereas caffeine induced exocytosis in a dose-dependent fashion. We then tested 45Ca2+ release also with isolated cell cortices (cell fragments containing cell membranes with stores and secretory organelles still attached). Under conditions which initiate exocytosis in vitro (depending on [ATP], reduction of [Mg2+] in presence of Ca2+; c.f. Lumpert et al. 1990, Biochem. J. 269, 639) we observed significant 45Ca2+ release with cortices as with isolated alveolar sacs. Our interpretation is as follows. (a) Alveolar sacs have a SERCA-type Ca(2+)-pump. (b) They have some sensitivity to caffeine, but none to ryanodine, InsP3 or cADPR. (c) There might be a direct functional coupling of these subplasmalemmal Ca2+ stores to the plasmalemma to which they are connected via feet-like structures; also like the SR, activation of this store is modulated by Mg2+ and ATP.

An exocytotic mutant of Paramecium caudatum: membrane fusion without secretory contents release.

This is a detailed characterization of a secretory mutant incapable of releasing secretory contents despite normal exocytotic membrane fusion performance. Trichocyst non-discharge strain tnd1 of Paramecium caudatum and its wildtype (wt) both show a transient cortical [Ca2+]i increase and exocytotic membrane fusion in response to the polyamine secretagogue, aminoethyldextran (AED), or to caffeine. tnd1 cells frequently display spontaneous Ca2+ signals parallelled by spontaneous exocytotic membrane fusion. This remains undetected, unless the trichocyst matrix is shown to be freely accessible to the inert, non-membrane permeable fluorochrome, F2FITC, from the outside. In these tnd1 cells, spontaneous and AED- or caffeine-induced membrane fusion, always without contents expulsion by decondensation (i.e. several-fold stretching), is ascertained by electron microscopy. Exocytotic openings, with condensed trichocysts retained, may persist for hours without impairing cells. Trichocyst decondensation normally requires micromolar [Ca2+]e, but an increase to 10 mM has no effect on tnd1 trichocyst expansion in vivo or in vitro (when isolated and exposed to ionophore A23187 + Ca2+). Paracrystalline packing of the major secretory components (trichynins) does occur, despite incomplete proteolytic precursor processing (according to SDS-PAGE). However, 45Ca(2+)-binding by secretory components is considerably reduced--the likely cause of the non-discharge phenotype. Our findings imply significant untriggered membrane fusion in a system normally following the triggered pathway and clear separation of exocytotic membrane fusion from any later Ca(2+)-dependent steps of the secretory cycle.

Effects of adrenergic agonists and antagonists on the numbers of synaptic ribbons in the rat pineal gland.

In the pineal gland numbers of synaptic ribbons (SR) undergo day/night changes which parallel the rhythm of melatonin synthesis. Since pineal biosynthetic activity is controlled by activation of adrenoreceptors, we investigated the effects of adrenergic agonists and antagonists on pineal synaptic ribbon numbers and N-acetyltransferase (NAT) activity, the key enzyme of melatonin synthesis in rats. In vivo application of the beta-adrenergic antagonist propranolol decreased melatonin synthesis when given during the dark phase but did not affect SR numbers. Treatment during daytime with the beta-adrenergic agonist isoproterenol increased pineal NAT activity whereas SR numbers did not change. Norepinephrine stimulated NAT activity in vitro in a dose-dependent manner, but did not elevate SR numbers. Incubation with an analog of the second messenger cyclic adenosine monophosphate increased both NAT activity and SR numbers. These results suggest that the beta-adrenergic system does not play a decisive role in the regulation of the nocturnal increase in SR numbers observed in the rat pineal gland.

Differential distribution of calcium stores in paramecium cells. Occurrence of a subplasmalemmal store with a calsequestrin-like protein.

We have analyzed in Paramecium cells the occurrence and intracellular distribution of the high capacity/low affinity calcium-binding proteins, calsequestrin (CS) and calreticulin (CR) using antibodies against CS from rat skeletal muscle and against CR from rat liver, respectively. As revealed by Western blots, a CS-like protein isolated by affinity chromatography from Paramecium cells comigrated with CS isolated from rat skeletal muscle. The immunoreactivity of this 53 kDa protein band was blocked when the antibodies had been preadsorbed with purified rat CS. A band of identical molecular size was shown to bind 45Ca in overlays. By immunofluorescence and immunogold labeling this CS-like protein was localized selectively to the extended subplasmalemmal calcium stores, the "alveolar sacs", which cover almost the entire cell surface. Concomitantly the 53 kDa 45Ca-binding band became increasingly intense in overlays as we increasingly enriched alveolar sacs. Antibodies against rat CR react with a 61 kDa band but do not cross-react with CS-like protein in Paramecium. These antibodies selectively stained intracellular reticular structures, identified bona fide as endoplasmic reticulum.

Permanent Genetic Resources added to the Molecular Ecology Resources Database 1 February 2010-31 March 2010.

This article documents the addition of 228 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Anser cygnoides, Apodemus flavicollis, Athene noctua, Cercis canadensis, Glis glis, Gubernatrix cristata, Haliotis tuberculata, Helianthus maximiliani, Laricobius nigrinus, Laricobius rubidus, Neoheligmonella granjoni, Nephrops norvegicus, Oenanthe javanica, Paramuricea clavata, Pyrrhura orcesi and Samanea saman. These loci were cross-tested on the following species: Apodemus sylvaticus, Laricobius laticollis and Laricobius osakensis (a proposed new species currently being described).

Spectroscopic probing of dynamic changes during stimulation and cell remodeling in the single cardiac myocyte.

Optical microscopy, involving both fluorescence imaging and confocal Raman microspectroscopy, was used to visualize single, isolated, electrically active heart muscle cells. For example, short-term, dynamic changes in Raman bands during the contraction cycle, as well as persistent band changes during structural remodeling (microscopic rearrangements of cellular structures) in culture over longer periods of time, were obtained from the cellular content (sarcoplasm) of the heart cells. The results of the short-term studies, collected during electrical stimulation, showed dynamic changes in the Raman amide I band intensity, which occurred in phase with changes in cell length during cardiomyocyte contraction. The longer term studies of quiescent cardiomyocytes in culture over 3 days revealed a progressive and sustained increase in the intensity of the amide I band. Over the same period of culture, a decrease in the number of t-tubules (invaginations of the cell membrane, sarcolemma, which ensure the spreading of the action potential into the bulk of the sarcoplasm) was observed using confocal z-sections of the fluorescently labeled sarcolemma. The ability to measure both short-term dynamic changes associated with stimulated contraction and longer term persistent remodeling in the structure of intracellular macromolecules is valuable for assessing the physiological state of the cell, in real time.

"Frustrated Exocytosis"--a novel phenomenon: membrane fusion without contents release, followed by detachment and reattachment of dense core vesicles in Paramecium cells.

The lipophilic fluorescent dye, FM1-43, as now frequently used to stain cell membranes and to monitor exo-endocytosis and membrane recycling, induces a cortical [Ca(2+)](i) transient and exocytosis of dense core vesicles ("trichocysts") in Paramecium cells, when applied at usual concentrations (

Imaging of Ca2+ transients induced in Paramecium cells by a polyamine secretagogue.

In Paramecium tetraurelia cells analysis of transient changes in Ca2+ concentration, [Ca2+]i, during aminoethyldextran (AED) stimulated synchronous (<1 second) trichocyst exocytosis has been hampered by various technical problems which we now have overcome. While Fura Red was found appropriate for quantitative double wavelength recordings, Fluo-3 allowed to follow, semi-quantitatively but with high time resolution, [Ca2+]i changes by rapid confocal laser scanning microscopy (CLSM). Resting values are between 50 and 70 nM in the strains analysed (7S wild type, as well as a non-discharge and a trichocyst-free mutant, nd9-28 degrees C and tl). In all strains [Ca2+]i first increases at the site of AED application, up to 10-fold above basal values, followed by a spillover into deeper cell regions. This might: (i) allow a vigorous Ca2+ flush during activation, and subsequently (ii) facilitate re-establishment of Ca2+ homeostasis within > or =20 seconds. Because of cell dislocation during vigorous trichocyst exocytosis, 7S cells could be reasonably analysed only by CLSM after Fluo-3 injection. In 7S cells cortical [Ca2+]i transients are strictly parallelled by trichocyst exocytosis, i.e. in the subsecond time range and precisely at the site of AED application. Injection of Ca2+ is a much less efficient trigger for exocytosis. Ca2+-buffer injections suggest a requirement of [Ca2+]i >1 to 10 microM for exocytosis to occur in response to AED. In conclusion, our data indicate: (i) correlation of cortical [Ca2+]i transients with exocytosis, as well as (ii) occurrence of a similar signal transduction mechanism in mutant cells where target structures may be defective or absent.

Caffeine-induced Ca2+ transients and exocytosis in Paramecium cells. A correlated Ca2+ imaging and quenched-flow/freeze-fracture analysis.

Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti approximately 50 to 80 nm, [Ca2+]acti rises within /=2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge.

Polyamine triggering of exocytosis in Paramecium involves an extracellular Ca(2+)/(polyvalent cation)-sensing receptor, subplasmalemmal Ca-store mobilization and store-operated Ca(2+)-influx via unspecific cation channels.

The polyamine secretagogue, aminoethyldextran (AED), causes a cortical [Ca(2+)] transient in Paramecium cells, as analyzed by fluorochrome imaging. Our most essential findings are: (i) Cortical Ca(2+) signals also occur when AED is applied in presence of the fast Ca(2+) chelator, BAPTA. (ii) Extracellular La(3+) application causes within seconds a rapid, reversible fluorescence signal whose reversibility can be attributed to a physiological [Ca(2+)](i) transient (while injected La(3+) causes a sustained fluorescence signal). (iii) Simply increasing [Ca(2+)](o) causes a similar rapid, short-lived [Ca(2+)](i) transient. All these phenomena, (i-iii), are compatible with activation of an extracellular "Ca(2+)/(polyvalent cation)-sensing receptor" known from some higher eukaryotic systems, where this sensor (responding to Ca(2+), La(3+) and some multiply charged cations) is linked to cortical calcium stores which, thus, are activated. In Paramecium, such subplasmalemmal stores ("alveolar sacs") are physically linked to the cell membrane and they can also be activated by the Ca(2+) releasing agent, 4-chloro-m-cresol, just like in Sarcoplasmic Reticulum. Since this drug causes a cortical Ca(2+) signal also in absence of Ca(2+)(o) we largely exclude a "Ca(2+)-induced Ca(2+) release" (CICR) mechanism. Our finding of increased cortical Ca(2+) signals after store depletion and re-addition of extracellular Ca(2+) can be explained by a "store-operated Ca(2+) influx" (SOC), i.e., a Ca(2+) influx superimposing store activation. AED stimulation in presence of Mn(2+)(o) causes fluorescence quenching in Fura-2 loaded cells, indicating involvement of unspecific cation channels. Such channels, known to occur in Paramecium, share some general characteristics of SOC-type Ca(2+) influx channels. In conclusion, we assume the following sequence of events during AED stimulated exocytosis: (i) activation of an extracellular Ca(2+)/polyamine-sensing receptor, (ii) release of Ca(2+) from subplasmalemmal stores, (iii) and Ca(2+) influx via unspecific cation channels. All three steps are required to produce a steep cortical [Ca(2+)] signal increase to a level required for full exocytosis activation. In addition, we show formation of [Ca(2+)] microdomains (

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels.

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mM veratridine by immediate trichocyst exocytosis, provided [Ca2+]o (extracellular Ca2+ concentration) is between about 10(-4) to 10(-3) M as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec. Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca2+o or increasing [Ca2+]o to 10(-2) M inhibit exocytotic membrane fusion. Veratridine does not release Ca2+ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na+o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca2+]o) in the somatic, i.e., nonciliary cell membrane and that a Ca2+ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.

Veratridine-mediated Ca2+ dynamics and exocytosis in paramecium cells.

We analyzed [Ca2+]i transients in Paramecium cells in response to veratridine for which we had previously established an agonist effect for trichocyst exocytosis (Erxleben & Plattner, 1994. J. Cell Biol. 127:935-945; Plattner et al., 1994. J. Membrane Biol. 158:197-208). Wild-type cells (7S), nondischarge strain nd9-28 degrees C and trichocyst-free strain "trichless" (tl), respectively, displayed similar, though somewhat diverging time course and plateau values of [Ca2+]i transients with moderate [Ca2+]o in the culture/assay fluid (50 microM or 1 mm). In 7S cells which are representative for a normal reaction, at [Ca2+]o = 30 nm (c.f. [Ca2+]resti = approximately 50 to 100 nm), veratridine produced only a small cortical [Ca2+]i transient. This increased in size and spatial distribution at [Ca2+]o = 50 microM of 1 mm. Interestingly with unusually high yet nontoxic [Ca2+]o = 10 mm, [Ca2+]i transients were much delayed and also reduced, as is trichocyst exocytosis. We interpret our results as follows. (i) With [Ca2+]o = 30 nm, the restricted residual response observed is due to Ca2+ mobilization from subplasmalemmal stores. (ii) With moderate [Ca2+]o = 50 microM to 1 mm, the established membrane labilizing effect of veratridine may activate not only subplasmalemmal stores but also Ca2+o influx from the medium via so far unidentified (anteriorly enriched) channels. Visibility of these phenomena is best in tl cells, where free docking sites allow for rapid Ca2+ spread, and least in 7S cells, whose perfectly assembled docking sites may "consume" a large part of the [Ca2+]i increase. (iii) With unusually high [Ca2+]o, mobilization of cortical stores and/or Ca2+o influx may be impeded by the known membrane stabilizing effect of Ca2+o counteracting the labilizing/channel activating effect of veratridine. (iv) We show these effects to be reversible, and, hence, not to be toxic side-effects, as confirmed by retention of injected calcein. (v) Finally, Mn2+ entry during veratridine stimulation, documented by Fura-2 fluorescence quenching, may indicate activation of unspecific Me2+ channels by veratridine. Our data have some bearing on analysis of other cells, notably neurons, whose response to veratridine is of particular and continuous interest.

Green fluorescent protein-tagged sarco(endo)plasmic reticulum Ca2+-ATPase overexpression in Paramecium cells: isoforms, subcellular localization, biogenesis of cortical calcium stores and functional aspects.

We have followed the time-dependent transfection of Paramecium cells with a vector containing the gene of green fluorescent protein (GFP) attached to the C-terminus of the PtSERCA1 gene. The outlines of alveolar sacs (ASs) are labelled, as is the endoplasmic reticulum (ER) throughout the cell. When GFP fluorescence is compared with previous anti-PtSERCA1 antibody labelling, the much wider distribution of GFP (ER+ASs) indicates that only a small amount of SERCA molecules is normally retained in the ER. A second isoform, PtSERCA2, also occurs and its C-terminal GFP-tagging results in the same distribution pattern. However, when GFP is inserted in the major cytoplasmic loop, PtSERCA1 and two fusion proteins are mostly retained in the ER, probably because of the presence of the overt C-terminal KKXX ER-retention signal and/or masking of a signal for transfer into ASs. On the overall cell surface, new SERCA molecules seem to be permanently delivered from the ER to ASs by vesicle transport, whereas in the fission zone of dividing cells ASs may form anew. In cells overexpressing PtSERCA1 (with C-terminal GFP) in ASs, [Ca2+]i regulation during exocytosis is not significantly different from controls, probably because their Ca2+ pump has to mediate only slow reuptake.

Functional and fluorochrome analysis of an exocytotic mutant yields evidence of store-operated Ca2+ influx in Paramecium.

A non-discharge mutant of Paramecium tetraurelia (nd12-35 degrees C, lacking exocytotic response upon stimulation with the nonpermeable polycationic secretagogue aminoethyldextran, AED), in the pawnA genetic context (d4-500r, lacking ciliary voltage-dependent Ca2+ influx), was shown to lack (45)Ca2+ entry from outside upon AED stimulation. In contrast, cells grown at 25 degrees C behave like the wildtype. To check the functional properties in more detail, fluorochrome-loaded 35 degrees C cells were stimulated, not only with AED (EC(100) = 10(-6) M in wildtype cells), but also with 4-chloro-meta-cresol, (4CmC, 0.5 mM), a permeable activator of ryanodine receptor-type Ca2+ release channels, usually at extracellular [Ca2+] of 50 microM, and eventually with a Ca2+ chelator added. We confirm that pwA-nd12(35 degrees C) cells lack any Ca2+ influx and any exocytosis of trichocysts in response to any stimulus. As we determined by x-ray microanalysis, total calcium content in alveolar sacs (subplasmalemmal stores) known to be mobilized upon exocytosis stimulation in wild-type cells, contain about the same total calcium in 35 degrees C as in 25 degrees C cells, and Ca2+ mobilization from alveoli by AED or 4CmC is also nearly the same. Due to the absence of any AED-induced Ca2+ influx in 35 degrees C cells and normal Ca2+ release from stores found by x-ray microanalysis one can exclude a "CICR"-type mechanism (Ca2+-induced Ca2+ release) and imply that normally a store-operated Ca2+ ("SOC") influx would occur (as in 25 degrees C cells). Furthermore, 35 degrees C cells display a significantly lower basal intracellular [Ca2+], so that any increase upon stimulation may be less expressed or even remain undetected. Under these conditions, any mobilization of Ca2+ from stores cannot compensate for the lack of Ca2+ influx, particularly since normally both components have to cooperate to achieve full exocytotic response. Also striking is our finding that 35 degrees C cells are unable to perform membrane fusion, as analyzed with the Ca2+ ionophore, A23187. These findings were corroborated by cryofixation and freeze-fracture analysis of trichocyst docking sites after AED or 4CmC stimulation, which also revealed no membrane fusion. In sum, in nd12 cells increased culture temperature entails multiple defects, notably insensitivity to any Ca2+ signal, which, moreover, cannot develop properly due to a lower basal [Ca2+] level and the lack of Ca2+ influx, despite normal store activation.

Immunolocalization of the exocytosis-sensitive phosphoprotein, PP63/parafusin, in Paramecium cells using antibodies against recombinant protein.

We have localized a structure-bound fraction of the exocytosis-sensitive phosphoprotein, PP63/parafusin (PP63/pf), in Paramecium cells by widely different methods. We combined cell fractionation, western blots, as well as light and electron microscopy (pre- and post-embedding immunolabeling), applying antibodies against the recombinant protein. PP63/pf is considerably enriched in certain cortical structures, notably the outlines of regular surface fields (kinetids), docking sites of secretory organelles (trichocysts) and the membranes of subplasmalemmal Ca2+-stores (alveolar sacs). From our localization studies we tentatively derive several potential functions for PP63/pf, including cell surface structuring, assembly of exocytosis sites, and/or Ca2+ homeostasis.

In vitro effects of putative neurotransmitters on synaptic ribbon numbers and N-acetyltransferase activity in the rat pineal gland.

The pineal contains a large number of classical transmitters and neuropeptides. Some of these neurochemicals are involved in the regulation of serotonin N-acetyltransferase (NAT) activity and hence in melatonin synthesis. Synaptic ribbons present in the pineal gland also exhibit a numerical day/night rhythm parallel to that of NAT activity. There is scarcity of information regarding the regulation of synaptic ribbon (SR) numbers. In the present study, we have investigated in vitro effects of a number of classical neurotransmitters and neuropeptides. NAT activity was used to monitor melatonin synthesis under the experimental conditions used. Norepinephrine (NE), Delta sleep-inducing peptide (DSIP), vasoactive intestinal polypeptide (VIP), adenosine and N-acetyl-asp-glu (NAAG) significantly increased NAT activity in rat pineal. DSIP and VIP also increase the stimulatory effect of NE on NAT activity. These neurochemicals had no effect on SR numbers. Gamma aminobutyric acid (GABA), serotonin and taurine affected neither NAT activity nor SR. Somatostatin increased SR numbers significantly, without having any effect on NAT activity. The effect of somatostatin is regarded to be pharmacologic, since rather high dosages (10(-4) M) were required to obtain a significant effect. Although somatostatin is present in the pineal and may change rhythmically, the inconsistency of the day/night rhythmicity and the lack of such a rhythm in female rats and male gerbils speaks against an important physiological role of somatostatin in regulating SR numbers.