Electrical properties and cell-to-cell communication of the salivary gland cells of the snail, Helix pomatia

Comparative Biochemistry and Physiology, Part A 145 (2006) 7 – 19 www.elsevier.com/locate/cbpa

Electrical properties and cell-to-cell communication of the salivary gland cells of the snail, Helix pomatia Zsolt Pirger, Károly Elekes, Tibor Kiss ⁎ Department of Experimental Zoology, Balaton Limnological Research Institute, Hungarian Academy of Sciences, Tihany, 8237, Klebelsberg K. u. 3., Hungary Received 11 November 2005; received in revised form 22 March 2006; accepted 24 March 2006 Available online 30 June 2006

Abstract The aim of the present study was to assess the cellular mechanism of secretion in the salivary gland of the snail, Helix pomatia, using electrophysiological, electron microscopic and immunohistochemical techniques. A homogeneously distributed membrane potential (−56.6 ± 9.8 mV) was determined mainly by a K+-electrochemical gradient and partly by the contribution of the electrogenic Na+-pump and Cl− conductance. Low resistance electrical coupling sites were identified physiologically. Transmission electron microscopy and innexin 2 antibody revealed the presence of gap–junction-like membrane structures between gland cells. It is suggested that gap–junctions are sites of electrotonic intercellular communication, which integrate the gland cells into a synchronized functional unit in the acinus. Stimulation of the salivary nerve elicited secretory potentials (depolarization) which could be mimicked by local application of acetylcholine, dopamine or serotonin. In voltage-clamp experiments four major conductances were identified: a delayed rectifier (IK), a transient (IA) and a Ca2+-activated outward K+ current (IK(Ca)) and Ca2+-inward currents (ICa). It is suggested that one or more of these conductances may give rise to a stimulus activated secretory potential leading to excitation–secretion coupling and subsequent the release of the mucus from the gland cells. © 2006 Elsevier Inc. All rights reserved. Keywords: Snail; Salivary gland; Ion-channels; K+-currents; Innexin; Gap–junction; Cell contacts; Secretion; Secretory potential

1. Introduction Glands releasing their secretory material on to the surface via a system of ducts are called exocrine glands. Their secretory units consist of acinar structures comprising grape-like clusters of gland cells. Exocrine glands discharge their products by merocrine, apocrine or holocrine type of secretion. In holocrine secretion, the entire cell is broken down during the release of secretory product. Recently, it became evident that the holocrine secretion is tightly regulated by the mechanisms called apoptosis (Wrobel et al., 2003). This type of secretion is typical for a number of exocrine glands in both vertebrates and invertebrates, such as sebaceous (Clarys and Barel, 1995), uropygial (Rongone et al., 1967; Suzuki et al., 1994), axillary (Cameron and Endean, 1971), harderian (Djeridane, 1994) and venom glands (Cameron and Endean, 1972; Malli et al., 2000). The exact release mechanism of the secretory product, however, is still controversial. ⁎ Corresponding author. Tel.: +36 87 448 244; fax: +36 87 448 006. E-mail address: [email protected] (T. Kiss). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.06.042

Gastropod molluscs are extensively used as models to study the cellular basis of discrete physiological functions (Beltz and Gelperin, 1979; Benjamin et al., 2000; Chase, 2000). Pulmonate gastropods generally possess a pair of salivary glands with a complex anatomical organization (Andrews, 1991; Voltzow, 1994). In the salivary glands of Helicids, for example, several authors described morphologically and ultrastructurally distinct cell types in the gland, whereas others were on the opinion that these cells represented various stages of a secretory cycle (Moreno et al., 1982; Charrier, 1988; Serrano et al., 1996). Recently, we have identified three different cell types in the salivary gland of Helix, which were not distributed evenly in the acinus, but rather they showed a tendency toward clustering. Furthermore, proliferating cells were observed at the periphery of the acini and apoptotic cells occurred close to intralobular ducts, suggesting the presence of a continuous renewal process or a “secretory cycle” in the salivary gland (Boer, 1967; Kater et al., 1978a,b; Pirger et al., 2004). In vertebrates the complex relationship between cell proliferation, differentiation and death is an important feature in maintaining the normal architecture and function of salivary gland tissue. It was shown that both acinar and ductal cells exhibit

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regulated proliferation and apoptosis in mice that assure appropriate cell survival and renewal cycle, a property characteristic for many exocrine glands (Actis et al., 2002). Salivary glands of gastropods are highly amenable to studies of the mechanisms of stimulus–secretion coupling, the neuronal regulation of glandular secretion and integration of this process into complex behavioural actions (Kater et al., 1978b). Morphological studies on molluscan salivary glands revealed a strikingly similar basic structure across different gastropod species (Moura et al., 2004). Gland cells are organized in acinar structures and the cells are facing with the luminal membrane inside the acinus and having a basal membrane outside the acinus. Glandular cells are thought to secrete a primary product (mucus), the composition of which could be modified meanwhile passing through the salivary duct (Beltz and Gelperin, 1979). Evidence was presented showing that salivary glands of gastropods are controlled by two electronically coupled buccal neurons (Kater et al., 1978a; Altrup and Speckmann, 1982). Further on, excitatory postsynaptic, miniature, resting and action potentials (AP) were recorded from glandular cells of different molluscan species (Kater et al., 1978a; Bahls et al., 1980; Barber, 1983; Copeland and Gelperin, 1983). It also was observed that responses to nerve stimulation could be mediated by acetylcholine, dopamine, glutamate or serotonin acting as neurotransmitter (s) or modulator(s) (Ginsborg and House, 1976; Barber, 1982; Bahls, 1987; Bahls et al., 1995; Kiss et al., 2003). Furthermore, applying a multiple-site optical recording system it was found that neuronal stimulation directly activated a regenerative gland response, while the excitation of distal gland regions was mediated by electrotonic spread from active glandular cells (Senseman et al., 1983). Electrotonic coupling between glandular cells within the acini was characterized physiologically in Helisoma (Kater et al., 1978b). It was reported that cell-to-cell interactions were essential for the coordinated functioning of gland cells. The morphological basis of cell-to-cell communication is a pentalaminar gap–junction like membrane structure, containing channels composed of proteins belonging to the connexin family (Lampe and Lau, 2000). Despite the rich information on the neuronal regulation of the gastropod salivary gland, there are no data either about the molecular nature of the gap–junction or on the ion-conductances underlying the electrical activity of these cells in resting state and during saliva production and secretion. Since apoptosis seems to be the physiological mechanism responsible for the saliva release from the snail salivary gland cells (Pirger et al., 2004), the characterization of the K+-channel subtypes of these cells could be an important issue. It is widely accepted that K+-homeostasis plays a key role in apoptosis. Compelling evidence now also indicates that voltage-gated K+-channels provide a pathway for pro-apoptotic K+-efflux (Yu, 2003). The present paper contains the results of a series of investigations, which was undertaken to describe the basic electrical properties of the snail salivary gland cells, focusing especially on the spread of the excitation between gland cells. Ultrastructural and immunohistochemical approaches were applied, in order to characterize the types of membrane contacts involved in cell-to-cell communication, including interglandular and neuro-glandular interactions. Further on, we measured and

characterized, for the first time, ion-currents present in the gland cells. Hence our data provide a basis for further experiments to explore details of the cellular mechanism of the saliva production and release in the snail salivary gland. 2. Materials and methods 2.1. Experimental animals and preparation Both physiological and morphological experiments were carried out on the salivary gland of adult specimens of Helix pomatia L. Animals were kept under wet condition in an aquarium at room temperature (24 ± 2 °C) and fed on lettuce or cucumber twice a week, so they remained in an active state. Dissection was performed as previously described (Kiss et al., 2003). In case of electrical stimulation, salivary glands attached to the buccal ganglia were used. Approximately 2–3-mm2 pieces of the anterior salivary gland were used for voltage-clamp experiments. Both whole salivary glands and pieces of the gland were placed in a Sylgard-coated recording chamber (total volume of approx. 2 mL and 0.4 mL), pinned out and perfused at a rate of 1–2 mL/min with normal physiological saline. 2.2. Electrophysiology Standard snail physiological solution was used in the experiments containing (in mM): NaCl 80, KCl 4, CaCl2 10, MgCl2 5 and TRIS-Cl 10, dissolved in distilled water and adjusted with NaOH to pH = 7.4. Inward calcium currents were studied in saline containing tetraethylammonium (TEA, 30 mM) and 4 aminopyridine (4-AP, 4 mM), in order to block potassium channels. Inward calcium currents were blocked by adding 100 μM CdCl2 to the above mentioned solution. In Cl− free solution acetate ions were substituted in equimolar amount for Cl−. 5-nitro-2-(3 phenylpropylamino)benzoate (NPPB) was used to block Cl− channels. The following substances were used in the experiments: acethylcholine (ACh,), 3 hydroxytyramine (dopamine, DA) and 5 hydroxytryptamine (serotonin, 5-HT), 10− 4M ouabain. Electrogenic Na-pump was studied using high K+-solution, which contained (in mM): NaCl 80, KCl 320, CaCl2 10, MgCl2 5 and TRIS-Cl 10, pH = 7.4. All substances listed above were purchased from Sigma. In experiments applying electrical stimulation the salivary glands were separated by vaseline gap from the rest of the preparation and soaked in a solution containing 800 mM glycerin for 2 h before the experiments. Thereafter, the whole salivary gland was placed into physiological solution. This procedure decreased the strength of the excitation–contraction coupling of muscle cells (Kiss, 1977) within the gland and facilitated the stable intracellular recording from gland cells upon nerve stimulation. The glycerin treatment had no effect on the electrical properties of the gland cells. The mean of the resting membrane potential (− 56.0 ± 9.2 mV, n = 19) was comparable with that of the control values. Single, square wave pulses of 5 V amplitudes and 100 ms durations were delivered by bipolar metal electrodes to the salivary nerve. Electrophysiological experiments were performed at room temperature (23–25 °C). In order to measure transmembrane

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ionic currents, one-microelectrode voltage-clamp technique was applied, using an Axoclamp 2B amplifier (Axon Instruments, USA). Voltage-clamp protocols were generated by the pClamp program (Axon Instruments, Inc., Union City, CA, USA).

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Recordings were filtered at 1 kHz using a Bessel filter, digitized (TL-1 Interface) and subsequently stored on hard disc. Intracellular recording electrodes were pulled from borosilicate glass (type M1B150F-3, WPI) using a vertical puller (David

Fig. 1. Resting membrane potentials (MP) recorded from salivary gland cells. A — Distribution of the MP values was bell-shaped and could be fitted with a single Gaussiancurve. The mean value was −56.6 mV (S.D. ± 9.8 mV, n = 483). B — The relationship between MP and electrode resistance saturated above 20 MΩ electrode resistance and could be fitted by single decaying exponential. C — The resting MP was plotted as a function of the log of the extracellular potassium concentration ([K]o). The fit of the linear part (between 40 and 320 mM) gave a slope of 41 mV/decade, which deviates from the pure K-diffusion potential (58 mV/decade). Number of measurements is given in brackets. D — Effect of Cl− free solution on the resting MP. The MP curve was an average of 6 experiments. The cells were depolarized by 20 mV and reversed after washing with normal physiological solution (horizontal arrow). The dashed line shows the resting MP measured in control solution (−55.4± 3.2 mV, n = 6). E — Effect of the Cl− channel blocker NPPB. Perfusion of the Cl− free solution evoked a depolarization (black trace). In the presence of 10− 4 M NPPB the Cl− free solution failed to evoke a depolarization (gray trace), instead a small transient hyperpolarization could be observed. Vertical arrowheads show the moment of a solution change. After washing the preparation with normal physiological solution (horizontal arrows) the recovery was complete. F — Contribution of an electrogenic Na+-pump to the MP. High K+-solution (320 mM) elicited a biphasic change of the MP first hyperpolarizing thereafter depolarizing the gland cell. Hyperpolarization evoked by high K+-solution was blocked by ouabain (10− 4 M). Horizontal arrows indicate the wash out of a high K+-solution and dashed line shows the 0 mV value of the membrane potential. Vertical arrow shows the beginning of a high K+-exposure.

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Kopf Instruments, model 730, USA). Glass micropipettes were filled with 2.5 mM KCl, having resistance usually between 15 and 20 MΩ. The Ag/AgCl bath electrode was placed in a small well containing 2.5 M KCl. The well was, in turn, connected to the recording chamber via an agar bridge. The preparation was continuously superfused with physiological solution at a flow rate of 1–2 mL/min. ACh, DA and 5HT were applied by a home-made microperfusion system. The system incorporated an electronically controlled, high quality solenoid valve, which allowed the precise regulation of the volume (in 10 μL steps) to be injected into the bathing solution. The 10 mL reservoir of the perfusion system contained the transmitter substance at 10− 3 M concentration. Thirty microlitres from this reservoir was added to the 0.4 mL recording chamber which was perfused continuously with physiological solution. The final concentration of the substances delivered by this way was not higher than 10− 4–10− 5 M. Data of electrophysiological traces were plotted and analyzed with Origin 6.07 software (Microcal Software Inc., USA). Data are presented as original records and as mean values ± S.E.M. P-values less than 0.05 were considered to be statistically significant. 2.3. Immunohistochemistry Salivary glands (n = 9) were dissected, pinned out in Sylgardcoated Petri dish and fixed overnight at 4 °C in 4% para-

formaldehyde diluted in 0.1 M phosphate buffer (PB, pH = 7.4). After washing in PB (2 × 5 min), series of 14 μm-thick cryostat sections were cut and processed for immuncytochemistry as follows: 1) PBS containing 0.25% TritonX-100 (PBS-TX) for 2 × 5 s; 2) anti-innexin2 antiserum (donation of Dr. Jane Davies, University of Sussex, England) diluted 1:500 for 24 h; 3) goatanti-rabbit IgG conjugated with FITC or TRITC (DAKO) diluted 1:40 for 1.5 h for cryostat sections and 5 h for Vibratome slices. All antisera were diluted in PBS-TX containing 0.25% bovine serum albumin (BSA) and the incubations were performed at room temperature. Preparations were mounted either in Fluoromount (DAKO) or in Vecta-Shield containing propidium-iodide (Vector), following which a red or orange staining of the cell nuclei could be seen. Negative control experiments were performed by substituting primary innexin2 antibody with 1% normal rabbit serum. No labelling could be observed following these experiments. Preparations were viewed in a Zeiss Axioplan microscope equipped with an appropriate filter set. 2.4. Electron microscopy Salivary glands (n = 9) were dissected and placed in a fixative solution containing 2.5% glutaraldehyde or a mixture of 2.5% glutaraldehyde and 1% paraformaldehyde diluted in 0.1 M PB. Fixation lasted overnight at 4 °C followed by several washing in PB, and then postfixation was undertaken for 1 h at 4 °C in 1% OSO4 diluted in 0.1 M Na-cacodylate. After dehydration in

Fig. 2. Ultrastructure of interglandular contacts in Helix salivary gland. A — Close (arrows) and loose (star) membrane contacts between two gland cells (GC1, GC2). B — Higher magnification view of close unspecialized membrane contacts (arrows) between two gland cells (GC1, GC2). C — Electron microscopic view of the peripheral region of two adjacent gland cells (GC1, GC2), contacting with long parallel membrane segments displaying increased electron density (arrows), which resembles desmosome-like (zonula addherens) structures. Insert: Enlarged view of a specialized membrane contact, indicated by arrow in C. Dense appositions along the membranes are shown by arrowheads. D — Higher magnification of a gap–junction-like contact (arrow) established between two gland cells (GC1, GC2) cytoplasm protrusions can be seen. Asterisk — intercellular space, rER — rough surface endoplasmic reticulum elements, G — Golgi-complex unit, m — mitochondrium, SD — saliva droplet.

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graded ethanol and propylene oxide, the materials were embedded in Araldite (Durcupan ACM, Fluka). During dehydration the tissue samples were blockstained for 30 min in 70% ethanol saturated with uranyl acetate. Ultrathin sections were cut on an LKB Novacut ultramicrotome, stained with lead citrate and viewed in a JEOL 1200EX electron microscope.

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3. Results 3.1. Electrical properties of the gland cell Upon inserting a microelectrode into a gland cell, membrane potentials (MP) in the range of − 30 and − 80 mV were recorded,

Fig. 3. Distribution of innexin2 immunoreactivity in the Helix salivary gland. A1. Immunofluorescence small dots (arrows) located around and nearby gland cells are seen. A2. The same section as in A1, but the background was reduced using PhotoShop 7.0 software, showing a similar distribution of innexin2 immunoreative elements between the gland cells (arrows in A1 and A2). B1-2. Two images of gland cells showing immunofluorescence small dots (arrows) clearly located around the gland cells, whereas their nucleus (dark center) and cytoplasm are free of labeling. B2 — Enlarged view of a gland cell displaying a pericellular localization of the innexin2 immunoreactive elements. Scale bars: 50 μm for A1 and A2, 20 μm for B1 and 40 μm for B2. C — Electrotonic spread of the voltage response between gland cells elicited by injection of 5, 6, 7 and 8 nA current pulses (minus and plus) at V1 electrode and recorded at V2. The distance between injecting (V1) and recording (V2) electrodes was 70 μm and the resting MP’s recorded were −65 mVand −62 mV, respectively. The value of coupling coefficient was 0.24 (V1/V2). The dashed line shows the 0 mV level, and the arrowhead shows the removing of V2 electrode from the cell. No stimulation artifacts can be observed.

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when the gland was bathed in physiological solution (Fig. 1A). Distribution of MPs recorded from 483 gland cells (96 animals) was fitted by a single Gaussian-curve with a mean value of − 56.6 ± 9.8 mV. Due to the presence of muscle cells in the salivary gland (SG), stable recording was hindered by spontaneous contractions. In order to obtain constant recordings of the resting MP, approximately 2–3 mm2 pieces were cut from the SG and used in the experiments. Under such conditions the recorded MP remained stable for relatively long periods (usually between 15 and 30 min, sometimes up to 60 min). Another important condition of the stable recording was the use of high resistance microelectrodes (> 20 MΩ). A non-linear relationship was obtained between the electrode resistance and the recorded MP saturating above 20 MΩ (Fig. 1B). The gland cell input resistance varied from 2 to 5.5 MΩ with an average value of 2.4 ± 0.4 MΩ (n = 10, mean ± S.E.M). As physiological solutions containing different K+-concentrations were perfused through the tissue chamber the MP changed in a manner shown in Fig. 1C. Accordingly, tenfold change in the extracellular K+ caused 41 mV change of the MP suggesting the contribution of other ions or pump mechanisms. Gland cells were extremely resistant against high extracellular K+-concentration ([K+]o), because at low concentrations (between 4 and 24 mM) the size of the depolarization (6 mV) was significantly smaller than expected value (40 mV). Removing or decreasing the extracellular Na+ concentration increased the resistance of the gland cell against high [K+]o (up to 40–90 mM). Furthermore, full recovery of the MP was obtained returning to normal physiological solution even after application of 320 mM K+solution. Complete replacement of Cl− in a physiological solution by impermeant anions such as acetate depolarized the gland cell membrane by 20 mV (Fig. 1D). The depolarizing effect of a Cl− free solution was eliminated by a Cl− channel blocker NPPB, suggesting the participation of Cl− in determining the MP (Fig. 1E). Exposing the gland cells to high (up to 180–320 mM) extracellular K+-solution often elicited a biphasic change of the MP. First the cell membrane was hyperpolarized, thereafter depolarized during the continuous exposition to this solution. The hyperpolarization was absent when 10− 4 M ouabain, a blocker of a Na+/K+–ATP-ase was added to a high K+-solution (Fig. 1F). The results suggest that although the MP was attributed mainly to the K+-distribution in a resting state, a contribution of the electrogen Na+-pump, Na+- and Cl− conductances should be considered especially under certain circumstances such as high [K+]o.

membrane segments separated by a space of 10–20 nm (Fig. 2B), or by longer parallel membrane segments displaying increased electron density, separated by widened intercellular space (∼ 60 nm) (Fig. 2C and insert). These membrane contacts identified as zonula adherens (desmosome-like) structures. Occasionally, along short membrane segments, tight membrane contacts could be identified, where the opposing membranes exhibited increased electron density and faced each other without a clear (2–4 nm) intercellular space (Fig. 2D). These structures were identified as gap–junction-like contacts. In order to characterize the junction proteins and localize the distribution of gap–junctions, a Drosophila innexin and connexin-36 antibodies (donation of Prof. Peter Somogyi, MRC Anatomical Neuropharmacology Unit, Oxford, England), were applied in fluorescence immunohistochemical experiments. In experiments with connexin-36 antibody no labeling was obtained. Following incubation with the anti-innexin2 antibody (Stebbings et al., 2000), immunoreactive small fluorescence dots could be observed located around the Helix SG cells (Fig. 3A1). After

3.2. Electrical coupling and synaptic input of the salivary gland cells The ultrastructural analysis of the SG revealed different types of membrane contacts, including both close and loose membrane appositions intermingling between the adjacent glandular cells (Fig. 2A). Loose contacts were characterized by widened extracellular space, the distance between the opposing gland cell membrane segments varying between 20 and 150 nm. Close membrane appositions were characterized either by unspecialized

Fig. 4. Intracellular potentials recorded from salivary gland cells. A — Records of spontaneous action potential like (junction potentials) discharges with amplitude of about 15–30 mV. B — Action potential-like discharges elicited from gland cells by injecting 20 mV depolarizing voltage pulse.

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reducing the background by using PhotoShop 7.0 software, it became evident that the innexin2 immunoreactivity was localized between the secretory cells (Fig. 3A2). When applying the antiinnexin2 antibody on cryostat sections obtained from locust (Locusta migratoria migratorioides) SG, the same pattern of immunolabeling could be observed (not shown). Mounting the immunolabeled sections of the Helix SG in Vecta-Shield, which labels the nuclei, it could be unequivocally demonstrated that innexin2 immunoractive elements were always localized along the cell surface, and never observed in the cytoplasm or the nucleus (Fig. 3B1 and B2).

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Inserting two microelectrodes into pairs of glandular cells demonstrated a good electrical coupling (coupling coefficient 0.24) irrespective from the direction of the injected current pulse (Fig. 3C). These results show that the close membrane appositions might be the sites of low resistance electrical contact between gland cells. Following microelectrode penetration, SG cells were usually electrically silent although miniature end-plate potentials (not shown) and bursts of excitatory postsynaptic potentials (EPSP) were occasionally present (Fig. 4A). Overshooting action potentials could never be recorded. EPSPs or action potential-

Fig. 5. A–D Effects of salivary nerve stimulation and local application of Ach, 5HT and DA on the MP. A — Depolarization evoked by single electric stimulus applied to the salivary nerve. Stimulation artifact is shown by arrow. The response of the gland cell was a depolarization about 30 mV of amplitude. B–D — Responses of salivary gland cells to the brief extracellular application of Ach (B), 5-HT (C) or DA (D) (all at 10− 3 M concentrations). Arrows show the time of application and the dashed line depicts the 0 mV MP level. E, F — Ultrastuctural aspects of the innervation of the salivary gland. E — An axon bundle (A) located between gland cells (GC), accompanied by a glia process (asterisk). Note the presence of granules and vesicles of different ultrastructure in some of the axon profiles (A′). F — A varicosity (T), containing clear vesicles and medium dense granules, is deeply embedded into a salivary gland cell (GC) and forms a close but unspecialized contact (arrows) with a gland cell. Note that the profile is almost entirely surrounded by a gland cell cytoplasmic protrusion (arrowheads). For E and F: SD — saliva droplet, asterisk — glia process, g — glia granule.

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contained only neurotubules and mitochondria, and partly also electron lucent and/or dense-core vesicles and granules. Varicosities embedded into the gland cells and/or surrounded by cytoplasmic processes were seen to form intimate neuroglandular contacts without bearing any membrane specialization (Fig. 5F). Specialized neuro-glandular synapses could not be detected in this study, supporting earlier electron microscopic immunocytochemical observations obtained on different peripheral tissues of Helix (Elekes and Ude, 1994; Elekes, 2000). It is suggested therefore that chemical signals from neurons to gland cells are transmitted by volume-transmission (Vizi, 1984). 3.3. Voltage-clamp experiments In order to study the ionic-currents of the SG cells, the single-electrode voltage-clamp technique was used. Time- and

Fig. 6. Family of ion-currents recorded from salivary gland cells and the current–voltage relationship. A — Outward currents evoked by a sequence of voltage pulses delivered between − 60 and +20 mV in 5 mV increments from a holding potential of − 60 mV. B — Delayed outward current (IK) component is blocked by about 60% (insert) when 30 mM TEA is present in the extracellular solution. C — Subtraction of the remaining currents after TEA block from the control curves revealed the TEA-sensitive currents. D — Steady-state current– voltage relationship of outward currents recorded in control solution (filled square), in the presence of 30 mM TEA (filled circle) and the TEA-sensitive component (filled triangle) obtained by subtraction of B from A.

like activity could be elicited by strong depolarization (40 mV) of the silent cell (Fig. 4B). Electrical stimulation of the salivary nerve elicited a depolarizing response of the SG cell up to 30 mVs (Fig. 5A). Similar depolarizing responses could be elicited by microperfusion onto the cell surface of ACh, 5-HT or DA which are supposed to be transmitters or modulators in the SG of Helix (Fig. 5B, C, and D). Responses were evoked by 4 s exposure of the gland cell to one of the solutions listed above. Concentrations of transmitter substances were not exactly determined, but were less than order of 10− 4–10− 5 M. The results suggest that the salivary nerve contains more than one excitatory transmitter because all three substances elicited a depolarization which was reminiscent to the secretory potential obtained by nerve stimulation (Fig. 5A). At ultrastructural level, axon bundles composed of different profiles and accompanied by glia processes were also detected running among gland cells (Fig. 5E). The axon profiles partly

Fig. 7. Characterization of a transient component (IA) of the outward current. A — The membrane potential was conditioned at −90 mV for 50 ms thereafter voltage steps of 250 ms were applied in 5 mV increments from −60 mV. Using this protocol a transient component could be isolated. B — The IA is effectively blocked by 4 mM 4-AP present in the external solution. Histogram in the insert shows the average block by 4-AP (55%). C — Subtraction of B from A curves resulting in a 4AP sensitive component of the outward current. D — Current–voltage relationship of the outward currents obtained in control solution (filled square), in the presence of 4-AP (filled circle) and the subtracted currents (filled triangle).

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(I–V) relationship obtained before and after TEA application, as well as after the subtraction, shows that the outward current is composed mainly of the delayed rectifier current (Fig. 6D). The I–V curve of the TEA-sensitive component was activated at − 45 ± 3 mV (n = 5) and rectified in outward direction consistently with the activation threshold level, described for the delayed outward current in many other invertebrate and vertebrate cells (Rudy, 1988). Transient outward currents could also be seen in gland cells provided that depolarizing voltage steps were preceded by conditioning hyperpolarization to −90 mV for 300 ms. Transient currents rose rapidly to a peak thereafter decayed exponentially during depolarization (Fig. 7A). The transient current could be effectively blocked by externally applied 4 mM 4-AP, resulting in a 55 ± 8% (n = 4) decrease of the current (Fig. 7B). In Fig. 7C, a family of current traces is presented after subtraction of currents recorded before and after 4-AP application. I–V relationship of the 4-AP sensitive component was activated at a threshold potential of around −40 mV (Fig. 7D).

Fig. 8. Ca2+-dependent component of the outward K+-current. A — Outward currents evoked by a sequence of voltage pulses delivered between −60 and +10 mV in 5 mV increments from a HP of −60 mV and recorded in physiological saline. B — Membrane currents recorded at low extracellular Ca2+ (1 mM). The amplitude of outward current is decreased. C — Subtraction of B from A results in a Ca-dependent component of the outward current. D — Average current–voltage relationship of currents recorded in control solution and currents obtained after subtraction shows that 27 ± 1.5% of the total outward currents is carried by Ca2+activated K+-channels.

voltage-dependent inward and outward membrane currents could be recorded after step depolarizations of voltage-clamped SG cells. Typical membrane currents recorded from a gland cell held at − 60 mV and depolarized in 5 mV steps to + 20 mV are presented in Fig. 6A. Depolarizing voltage pulses elicited an outward current which reached its steady state in 30 ms and remained constant during a 300 ms pulse. In addition to the outward current an inward component was also present in several cells (see later, Fig. 9). The non-inactivating outward current component could be blocked by 30 mM TEA added to the external solution, resulting in a 61 ± 2% (n = 5, insert) decrease of the outward current (Fig. 6B). The TEA-sensitive component, obtained by digital subtraction of the currents before and after TEA application, first peaked and then declined to a steady state level (Fig. 6C) and resembled the delayed rectifier current common to many cell types (Hille, 1992). The current remaining after TEA-block suggests that the outward current has several components (Fig. 6B). The current–voltage

Fig. 9. Identification of the voltage-activated inward current of the gland cell. A — Families of inward and outward currents are shown. B — After blocking outward component by 30 mM TEA and 4 mM 4-AP the inward component is remained. C — The inward component was effectively blocked by 100 mM CdCl2 when added to the physiological saline. D — The mean current–voltage relationship obtained from n=11 cells shows that the inward current was activated from −30±5 mV, peaked at 0–10 mV and reversed at +30 mV. Holding potential was set to −60 mV.

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A significant fraction of the outward current was relatively insensitive to TEA (Fig. 6B). Since part of the outward currents was blocked by decreasing the concentration of Ca2+ in the external solution, it is suggested that this fraction of the outward current is a Ca2+-dependent K+-current (IK(Ca)). Fig. 8A shows a family of currents recorded in a physiological saline containing 10 mM Ca 2+ . Reducing the Ca 2+ -concentration in the extracellular solution to 1 mM caused a decrease of the total outward currents recorded (Fig. 8B). Subtracting the currents recorded in the presence of 1 mM Ca2+ from those recorded in 10 mM Ca2+ revealed the Ca2+-dependent component of the total outward current (Fig. 8C). The contribution of a IK(Ca) to the total outward current was found to be 27 ± 5% (n = 4, insert Fig. 8C). The mean I–V curve revealed that IK(Ca) component was activated at around − 30 mV and the potential dependence was similar to that of the delayed rectifier component (Fig. 8D). As it was already mentioned, it was possible to record inward component from several gland cells in addition to the outward currents. Currents in Fig. 9A were recorded in response to depolarizing voltage steps from − 60 mV holding potential. When the SG was bathed in normal physiological solution, both inward and outward currents were recorded. The inward current could be dissected when blocking outward currents by adding TEA (30 mM) and 4AP (4 mM) to the extracellular solution (Fig. 9B). The inward current was eliminated by a solution containing 100 μM Cd2+, indicating that the current was carried by Ca2+ (Fig. 9C). The average I–V characteristic revealed that this current was activated at − 35 mV, peaked at around + 10 mV and reached zero current level at + 30 mV (Fig. 9D). 4. Discussion In the present study, the electrophysiological properties of the SG cells of the snail H. pomatia were investigated, and these studies were extended by applying for first time voltage-clamp technique. Electrophysiological experiments were coupled with the ultrastructural analysis of the possible forms of interglandular and neuroglandular connections and immunocytochemistry was also used to visualize the sites of the interglandular gap– junctions. Our results contribute to the understanding of the mechanisms and regulation of the secretion release from exocrine glands. 4.1. Resting membrane and action potentials of gland cells The mean of the MP recorded from altogether 483 cells was − 56.6 ± 9.8 mV and showed a normal distribution, suggesting that differentiation between the gland cell types is not possible on the basis of the MP. However, a close correlation between the electrode resistance and the measured MP could be observed. The MP increased up to 20 MΩ of the electrode resistance thereafter remained constant. The calculated mean value of the MP in Helix glandular cells is lower than that found in Helisoma (Kater et al., 1978a). The stable microelectrode recording, which is a prerequisite of voltage-clamp experiments, ensured that penetrations were successful, and the recorded MP values were not artifacts due to the damaged membranes. The MP was

mainly determined by the distribution of K + across the membrane, however, it deviated from the pure K+-diffusion potential. Similar deviation from the pure K+-diffusion potential was observed on the Helisoma SG cells (Hadley et al., 1980). The degree of deviation from electrodiffusion behaviour reflects the contribution of electrogenic transport, as well as other factors which can be influenced by changing the [K+]o. The depolarizing effect of Cl− free solution on the SG cell suggests the participation of Cl− in determining the MP. In this respect the Helix gland cell differs from those of Helisoma, in which Cl− did not contribute to the resting MP (Hadley et al., 1980). An interesting observation of our study was that the MP proved relatively insensitive to increasing [K+]o (up to 24 mM in normal physiological solution and up to 80 mM in Na-free solution), if the ionic and metabolic effects of changing [K+]o were approximately balanced. When this balance was disturbed the high K-solution exerted a biphasic effect first hyperpolarizing the glandular cell membrane by activating the electrogenic Na+pump and thereafter depolarizing it. Under these circumstances, both the increased intracellular Na+ and the increased [K+]o stimulated the Na+/K+ pump, which compensated the K-depolarization. Furthermore, the high [K+]o forced Cl− to move intracellularly, which again decreased the K-depolarization. The resistance against high K+-solution might also be explained by the special function and ion transport properties of gland cells: the basal and the luminal membranes have different properties (Bundgaard et al., 1977). The hyperpolarizing phase could be blocked by ouabain, indicating that the hyperpolarization was due to the activation of an electrogenic Na+-pump. Electrogenic Na+-pump might be involved in the regulation of the secretory process, similarly to other invertebrates and vertebrates, in which it was shown that secretion was dependent on the activation of Na+-K+-ATP-ases to maintain an inwardly directed Na+-gradient and intracellular K+-concentration (Hadley et al., 1980; Barber, 1985; Petersen, 1988; Mollard and Schlegel, 1996). On mammalian exocrine acinar tissue evidence was provided for the operation of a neutral Na+, K+, 2Cl− cotransport mechanisms, together with K+-channels and the Na+/K+ pump (Petersen, 1988). Whether such mechanism is present in the snail SG cells awaits for further examinations. The glandular cells of the SG of Helix exhibited action potentials usually not exceeding the resting MP (there was no overshoot). The capability of molluscan SG cells of producing action potentials is well documented, although in this property they differ from mammalian and insect exocrine gland cells. Kater et al. (1978a) investigated SGs from nine genera of gastropod molluscs and they concluded that action potential production in the gland cells is a general property among gastropods. Impulse activity was demonstrated in several mammalian gland cells as well, such as the cells of the intermediate lobe of pituitary (endocrine gland) and the insulinsecreting beta cells (exocrine gland) of the pancreas (Matthews and Sakamoto, 1975; Adler et al., 1983). The physiological significance of the impulse activity in gland cells is not fully understood. It can be suggested that its major function is to increase the cytosolic Ca2+-concentration and to synchronize the function of gland cell population within the acinus.

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4.2. Secretory potentials of gland cells In the Helix SG cells stimulation of the salivary nerve elicited a relatively slow, transient depolarization. Depolarizing secretory potentials could also be elicited in snail SG cells by local application either of ACh, 5-HT or DA. These data suggest that ACh, 5-HTand DA might be released from nerve endings upon the stimulation of the salivary nerve and could function as transmitters or modulators in saliva production and release. The possible neurotransmitter role of DA and 5-HT was already shown at the neuromuscular contacts of the salivary duct muscle cells (Kiss et al., 2003). The form of the depolarization wave was similar to the excitatory postsynaptic potentials observed in the SG cells of other molluscan species (Kater et al., 1978a; Bahls et al., 1980; Barber, 1982; Bahls et al., 1995), but its time course was much slower than that of the spontaneously occurring action potentials. Contrary, neural stimulation or local ACh application led to hyperpolarization in mammalian and insect SG cells (Lundberg, 1956; Ginsborg et al., 1974; Kagayama and Nishiyama, 1974). This hyperpolarization of acinar cell was called secretory potential (Imai, 1976). However, depolarizing secretory potential could be also elicited by applying ACh or field stimulation in mammalian exocrine pancreatic cells (Iwatsuki and Petersen, 1977; Davidson and Pearson, 1979). In our present study true neuro-glandular synapses were not found in the snail SG, but non-synaptic, close membrane contacts established by vesicle and/or granule containing varicosities occurred regularly, suggesting that the chemical (transmitter and/ or modulatory) regulation of the saliva release was realized by volume transmission (Elekes, 2000). 4.3. Intercellular contacts in the Helix SG In Helix, SG cells are organized in acinar units, similarly to that described earlier in other gastropods (Boer, 1967; Walker, 1970; Moreno et al., 1982; Bahls et al., 1995) and different exocrine glands of vertebrates (Shear, 1966; De Lange and Vreugdenhil, 1981; Stephens et al., 1987). The ultrastructural analysis of the gland, performed in the present study, shows that adjacent gland cells in the acini were coupled by at least two types of membrane contacts revealing specialization: i) desmosome-like (zonula adherend) occurring frequently, and ii) gap–junction-like tight membrane appositions. Immunohistochemical demonstration of a gap–junction protein, innexin2, of invertebrate origin (Stebbings et al., 2000) along the gland cell membranes speaks also for the presence of gap–junctions established between salivary gland cells in Helix. Electrophysiological recording from gland cells revealed a good coupling coefficient between them providing further evidence for the occurrence of the gap–junctions. Gap– junctions may have an important function in synchronising excitation of and subsequent release the saliva from the gland cells. 4.4. Ion-currents and their possible physiological significance in salivation Four voltage-gated currents in salivary gland cells of the snail were observed. Three of these currents were outward, carried by K+ and they differed from each other in kinetics and

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pharmacological properties. The fourth voltage-gated current was inward carried by Ca2+. Addition of these currents results in the total current waveform. The non-inactivating component (IK) was more sensitive to TEA and could be blocked at a concentration similar to that used for other molluscan muscle cells and neurons (Thompson, 1977; Klein and Kandel, 1978; Dorsett and Evans, 1989; Dorsett and Evans, 1991; Brezina et al., 1994). The transient outward current (IA) is more sensitive against the 4-AP block and less sensitive against the TEA. Separation of the two components, IK and IA, was facilitated by the voltage-dependent activation of the IA current which required a conditioning hyperpolarization. Several lines of evidence indicate that IK(Ca) contributed to the total current, because the outward current was attenuated either in low Ca2+solution or in the presence of the Ca2+-channel blocker Cd2+. In many exocrine cells both inward Ca2+-current (ICa) and IK(Ca) were shown to play a pivotal role in Ca2+-dependent mechanisms of the secretion (Petersen, 1988). Furthermore an ICa underlying spontaneous action potential also contributed to the synchronization of the cells in the acinus. Potassium channels are exceptionally diverse and single cells normally express several types of them in both excitable and non-excitable cells. They participate in a number of important cellular functions such as regulation of MP, muscle contraction, signal transduction, cell volume, cell proliferation and immune response, release of neurotransmitters, secretion of hormones and fluids (Hille, 1992). We suggest therefore that the physiological significance of membrane currents, beyond their usual functions, such as maintaining the resting MP, repolarizing the action potentials etc., comprises the regulation of saliva secretion and cell volume. At the resting MP the contribution of the delayed and IK(Ca) is almost 100%. The importance of Ca2+ and voltageactivated K+-channels was demonstrated in the basal membranes of several acinar cells from mammalian SG. Further on it was shown that K+-channels are functional parts of important pump mechanisms (Petersen, 1988). At hyperpolarized MP transient outward channels produced currents that were designed for high-frequency repetitive firing and generation of synchronization between cells as it was shown for discrete brain areas (Rudy and McBain, 2001). Three main mechanisms of the secretion are known, depending on the way how the secretory products are released from gland cells. In the merocrine mechanism, the content of the secretory vesicle leaves the cell by exocytosis, involving membrane fusion between the vesicle membrane and plasma membrane. In the process of apocrine secretion the secretory product is discharged along with parts of the apical cytoplasma. In the holocrine secretory mechanisms, the secretion product is released in the course of the destruction of the secretion-filled cell. In the SG of Biomphalaria two, apocrine and holocrine mechanisms, were detected by Moura et al. (2004). Recently, we have observed that in the SG of active (feeding) snail the number of apoptotic cells was increased, suggesting that a holocrine release was performed by apoptosis (Pirger et al., 2004). In the past years a significant body of evidence has accumulated demonstrating the role of voltage-dependent Ca2+-, K+- and Cl−

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channels in apoptosis. It was shown that cellular Ca2+-overload is a final common pathway leading to the cell death (Yu, 2003). Recent studies of several research groups suggest that changes in ionic, primarily K+, content play a key role in the progression of apoptosis. Enhancement of the plasma membrane K+-permeability was shown to be associated with an early response to apoptotic stimuli in a number of cell types (Bortner and Cidlowski, 1999; Dallaporta et al., 1999; Hribar et al., 2004). For example, it was found that delayed rectifier K+-channel was up-regulated during certain stages of different apoptotic stimuli in various cell types (Yu et al., 1997; Colom et al., 1998; Yu et al., 1998; Wang et al., 1999). On the other hand, TEA an effective blocker of delayed rectifier K+-channel attenuated significantly the apoptotic process in cortical neurons (Wang et al., 2000). In order to define the precise role of different ion-channels and Na-pump, separately and/or in concert, in determining the resting MP, the secretory potential and the saliva release and production further experiments are in progress. Acknowledgements This work was supported by OTKA grants No. 43216 (T.K.), and Nos., 34106 and 49090 (K.E). The skilful technical assistance of Ms. Zita László and Ms. Zsuzsa N. Fekete is greatly appreciated. Anti-innexin2 antiserum was a generous gift by Dr. Jane Davies (University of Sussex, England), and the connexin36 antiserum was donated by Prof. Peter Somogyi (MRC Anatomical Neuropharmacology Unit, Oxford, England). The authors are indebted to Dr. Zita Puskár (Semmelveis University, I. Institute of Anatomy, Budapest) for the technical support, and to Prof. G. Kemenes (University of Sussex, England) for the critical reading the manuscript. References Actis, A.B., Lampe, P.D., Eynard, A.R., 2002. Cellular basis and clinical implications of biological markers in salivary tissues: their topological distribution in murine submandibular gland. Oral Oncol. 38, 441–449. Adler, M., Wong, B.S., Sabol, S.L., Busis, N., Jackson, M.B., Weight, F.F., 1983. Action-potentials and membrane ion channels in clonal anteriorpituitary cells. Proc. Natl. Acad. Sci. U. S. A. 80, 2086–2090. Altrup, U., Speckmann, E.J., 1982. Responses of identified neurons in the buccal ganglia of Helix pomatia to stimulation of ganglionic nerves. Comp. Biochem. Physiol., A 72, 643–657. Andrews, E.B., 1991. The fine structure and function of the salivary glands of Nucella lapillus (Gastropoda: Muriciade). J. Molluscan Stud. 57, 111–126. Bahls, F., 1987. Acetylcholine-induced responses in the salivary gland cells of Helisoma trivolvis. Cell. Mol. Neurobiol. 7, 35–47. Bahls, F., Kater, S.B., Joyner, R.W., 1980. Neuronal mechanisms for bilateral coordination of salivary gland activity in Helisoma. J. Neurobiol. 11, 365–379. Bahls, F.H., Emery, D.G., Haydon, P.G., 1995. Glutamate-mediated synaptic transmission between neuron B4 and salivary cells of Helisoma trivolvis. Invertebr. Neurosci. 1, 123–131. Barber, A., 1982. Electrical responses of salivary-gland cells of the gastropod mollusk Philine aperta to putative neurotransmitters. 1. Acetylcholine and cholinergic agonists. Comp. Biochem. Physiol., C 73, 85–89. Barber, A., 1983. Nervous control of the salivary glands of the carnivorous mollusc Philine aperta. J. Exp. Biol. 107, 331–348. Barber, A., 1985. Actions of acetylcholine on the salivary gland cells of the pond snail, Planorbis corneus. Comp. Biochem. Physiol., C 80, 175–184.

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