Calcium-Dependent Action Potentials in Leech Giant Salivary Cells

To understand fully the phenomena involved in stimulus-secretion coupling (e.g. Ginsborg &,House, 1980), it will be necessary both to describe the membrane properties which lead to changes in membrane potential, and to elucidate the relationship between electrical and secretory events. Gland cell electrophysiology has often been technically difficult, due to the relatively small size of secretory cells which have been studied, and the electrotonic intercoupling within acinar structures (Petersen, 1980). Invertebrate animals have often provided useful preparations for the electrophysiological study of cellular phenomena of general interest, (Hodgkin & Huxley, 1952a; Bullock & Hagiwara, 1957; Hagiwara & Naka, 1964) because of experimentally favourable size, structure or specialization. Kater (1977) has noted the possible advantages of studying large molluscan exocrine cells, although these too are coupled, and pose problems in the interpretation of the electrical activity of single cells.

We report here that the giant salivary cells in Glossiphoniid leeches are exceptionally large, electrically and morphologically independent, and fire calcium-dependent action potentials.

Haementeria ghilianii were obtained from a breeding colony, and Haementeria officinalis collected from Lake Patzcuaro, Mexico, via Mr Joel Glover and the laboratory of Dr W. B. Kristan, University of California, San Diego. The animals were maintained at room temperature, 19–25 °C, in a holding solution of the following composition (mmol l⁻¹): NaCl, 4; K₂SO₄, 0·5; CaCl₂, 0·4; MgCl₂, 1; NaHCO₃, 2·4; Na₂CO₃, 0·3, at a pH between 8·75 and 9·0, approximating conditions in the lake from which they were obtained. No more than ten specimens were held in each 2-litre aquarium, and the solution was changed daily.

The leech was pinned to a wax block through its anterior and posterior suckers, and stretched to approximately its normal extended length. A dorsal midline incision was made through the external muscle layers of the anterior third of the animal, taking care not to cut too deeply. Reflecting the skin flaps and underlying connective tissue layers reveals two pairs of salivary glands attached to the base of the muscular proboscis (Fig. 1).

The anterior gland is isolated by freeing the distal end from the body wall by cutting the adhering connective tissue strands, and removing a small piece of the base of the proboscis with the proximal end attached. Removal of the proximal end is aided by cutting through the retractor muscle fibres attached to the smaller posterior glands, thus allowing the proboscis to move anteriorly and to straighten. An incision can then be made from the crop through the bulbous base of the proboscis, and a convenient small slip of muscle left attached to the proximal end of the anterior gland.

Dissection and electrophysiological recording were carried out in a Ringer’s solution (pH7·4) of the following composition (mmol l⁻¹): NaCl, 126; KC1, 4; CaCl₂, 2; Tris (Sigma Chemical), 10. All experiments were performed at ambient temperatures between 21 and 24°C.

The isolated anterior gland was pinned out using 0·15 mm minuten nadeln through the muscle slip at the proximal end and through either adhering strands of connective tissue or the gland itself at the distal end. Careful pinning is essential for obtaining stable recordings, and it is particularly important to stretch the gland to its original length, thereby re-applying tension to the investing connective tissue sheath. Despite the large size of the cell bodies, it sometimes proves difficult to insert a second microelectrode, as the cells are inclined to move within the gland when impalement is attempted.

The recording chamber was fashioned from a 7·5 × 2·5 × 0·8 cm Perspex block glued to a microscope slide, with a 3·5 × 1 cm slot drilled out and lined with Sylgard (Dow-Corning) to accommodate the gland. The bathing solution was exchanged by gravity feed and vacuum aspiration, and required 45 s to replace each chamber volume of 1·5 ml.

Cells were impaled with glass microelectrodes filled with 3 mol l⁻¹ KCl (resistance 30–45 MΩ), connected to a high input impedance amplifier. Cells were impaled under visual control using a Wild dissection microscope and illuminated by a sub stage darkfield condenser. Current was injected through the recording electrode using a bridge circuit, or via a second electrode inserted into the cell. All quantitative I/V measurements were made using separate current and voltage electrodes. The amplifier output was displayed on a Tektronix D-13 analogue storage oscilloscope, and permanent records obtained on Polaroid film or with an ink-writing oscillograph (Hewlett-Packard 7404A). Some records were stored on magnetic discs in a Nicolet 2090-III digital storage oscilloscope, and traced directly onto paper using an X-Y plotter.

Micropipettes were filled with Lucifer Yellow CH (gift of W. W. Stewart) dissolved in distilled water (5–10 mg ml⁻¹), and connected via a silver wire to the input stage of the amplifier. Dye was iontophoretically injected into cells by passing hyperpolarizing current pulses, 500 ms, 10 nA, once per second for 20–45 min. Successful dye injection produced a very pale yellow tint in the gland cell body, and was confirmed under the fluorescence microscope before processing the tissue.

The gland was fixed in 10% formalin for 30 min following overnight diffusion, dehydrated, cleared in xylene and mounted whole in light paraffin oil. The preparation was viewed using a Leitz MPV-2 microscope, and photographed using the attached Vario-Orthomat camera.

Inspection of the anterior gland under the dissection or compound microscope shows it to be composed of around 200 spherical or ovoid bodies (Fig. 2A). Each cell extends a single process, or ductule, anteriorly to the proboscis. The cytoplasm has obvious granular inclusions, and often appears more opaque at the side further from the ductule, probably because the secretory material displaces the nucleus to that side. The processes are narrow and grouped together where they enter the base of the proboscis (Fig. 2B). Some of them can be traced along their length following vital staining with Neutral Red dye, and terminate at various points along the proboscis, extending several centimetres from their respective cell bodies. In the related Rhyn-chobdellid leech Glossiphonia complanata, Damas (1962) has shown that the ductule terminals are distributed along the length of the proboscis, and exit variously into the cavity of the proboscis sheath, along the length of the proboscis lumen, and at the proboscis tip.

It is quite clear that each body is a single salivary gland cell. There is no acinar structure of any kind, nor any common duct: each cell has an individual ductule. These observations are strikingly confirmed by intracellular injection of the fluorescent dye molecule Lucifer Yellow CH into individual gland cell bodies (cf. Iwatsuki & Petersen, 1979). Fluorescence can be seen to be completely localized to the single cell body into which dye was injected (Fig. 3A), and this lack of dye-coupling excludes the possibility of cytoplasmic continuity between cells by means of the usual type of gap junction (Stewart, 1981). A single stained ductule may be followed along the length of the gland and into the proboscis in whole mount (Fig. 3B,C). Background illumination with white light allows the fluorescent process to be seen clearly among unstained ductules, and confirms there is no dye coupling where the ductules run in parallel at the proboscis base. In a recent histological investigation of these salivary cells, Sawyer, Damas & Tomic (1982) have made sections along the proboscis, and show that fluorescent dye is confined to single ductules at this level, and that there is no transport between adjacent processes.

Intracellular recording shows that cells in the isolated anterior gland are not spontaneously active, and a sample of 25 cells impaled with one microelectrode gives an average resting potential of −61 mV (S.D. 8 mV). A sample of 150 cells impaled with two microelectrodes over the course of many separate experiments gives an average resting potential of −50 mV (S.D. 11·6 mV). The more negative values tend to correspond to those impalements qualitatively judged to be better. Namely, a rapid negative-going deflection is obtained on the first attempt to impale the cell, a single action potential occurs, and the resting potential stabilizes within 1 min to a value which does not decay during the course of prolonged experimental manipulation.

At high external K⁺ concentrations, the values for resting potential conform closely to the slope predicted by the Nernst equation for a potassium equilibrium potential (Fig. 4). There is a clear deviation from the predicted slope at physiological K⁺ concentrations. Zero sodium solution does not alter either the resting potential or cell input resistance. The effect of varying external chloride has not been studied.

Cell input resistance varies with cell size : lower values for input resistance correlate with cells of larger diameter. We have found values of 5–16 MΩ corresponding to cell diameters in the range 400 to 80 µm. There is no correlation between input resistance and membrane potential, besides the observation that low input resistances of less than 1 MΩ are measured in cells where the resting potential has decayed to a low value, both resulting from the cell membrane being made leaky around the site of impalement.

Calculations based on a spherical cell body of diameter 300 μm and input resistance 6 MΩ yield an area-specific membrane resistance, Rm, of 17 kΩcm². Since this figure neglects the cell process and is a conservative estimate of cell surface area, it is an underestimate of Rm. Area-specific membrane capacitance, Cm, is generally regarded as a constant in animal cell membranes, having a value of approximately 1 μF cm^-2. This predicts a membrane time constant of 17 ms, whereas a measured value of 250 ms is obtained. Although this discrepancy might largely be accounted for by the underestimate of Rm, it is likely that the presence of the ductule influences the time course of membrane charging.

Recording from many pairs of adjacent and distant salivary cells clearly shows that they are not electrically coupled, that is, passing current pulses into one cell has no effect on the membrane potential of any other cell, consistent with the preceding anatomical observations.

We noted that the salivary cells often fire a single action potential in response to the depolarization resulting from impalement, but are subsequently silent. Depolarizing current pulses also elicit overshooting all-or-none action potentials, which last 100–200ms (Fig. 5A). The maximum peak-to-peak amplitude is 100mV, and the maximum rate of rise 5 V s⁻¹. The mean overshoot from 30 cells impaled with two microelectrodes is +17 mV, and both the rate of rise and the overshoot decrease as a cell becomes depolarized. Repolarization clearly occurs in two phases, followed by a pronounced after-hyperpolarization of long duration. Subthreshold current pulses can produce a voltage response which decreases with time, which is indicative of some form of delayed rectification (Hodgkin, Huxley & Katz, 1952).

Action potentials may also be elicited at the end of a hyperpolarizing pulse, so-called anode break excitation (Fig. 5B). An identical impulse is then seen, showing over shoot, shoulder on the repolarizing phase, and after hyperpolarization. This type of excitation has been rationalized on the basis of removal of voltage-sensitive sodium inactivation by the hyperpolarizing pulse in squid axon (Hodgkin & Huxley, 1952b), and may indicate the presence of voltage-dependent inactivation of calcium channels in the salivary cell membrane. However, measurement of ionic currents, such as Fox (1981) has demonstrated in eggs of the polychaete Neanthes, is clearly required before a definite statement can be made. No exogenous current is being applied during the impulse, and thus anode break is a convenient method of evoking action potentials when dual microelectrode impalement proves difficult, or when the voltage artefact caused by passing depolarizing current down a single recording electrode is unacceptably large.

The overshooting action potential is abolished when either 5 mmol l⁻¹ cobalt or 5 mmol l⁻¹ manganese is added to the Ringer’s solution (Fig. 6). As these solutions are washed in, the overshoot is reduced and the action potential is prolonged. A delayed rectification is seen when depolarizing current is applied once full blockade is achieved. Neither increased depolarizing current nor large hyperpolarizing currents result in a regenerative response under these conditions. The effect is completely reversible, and the same prolonged impulse of reduced amplitude may be seen before full recovery in normal Ringer’s. Lanthanum (La³⁺) is similarly effective in blocking the action potential at a lower concentration of 1 mmol l⁻¹ ; however, its action is not reversible.

There is no change in either the rate of rise or the overshoot when a preparation is exposed to sodium-free (arginine) Ringer’s solution (Fig. 7A). These impulse properties are not affected by tetrodotoxin (TTX) at a concentration of 2 × 10⁻⁵mol l⁻¹. These data provide substantive evidence that sodium current is not required for regenerative activity in these cells.

Action potentials persist when the external calcium is replaced by either strontium or barium (Fig. 7B). There is some prolongation of the action potential, and excitability is increased. Increased excitability as a result of lowering external calcium is well documented (e.g. Junge, 1981). This may be a result of a shift in the voltage sensitivity of inward current due to decreased screening of negative surface charges in zero-calcium solution. It may also be explained by a decreased calcium-sensitive potassium conductance, since there is no longer calcium influx during the action potential. Trains of impulses are seen in response to a single brief stimulus, whereas only a single action potential is elicited in normal solution.

When 2 mmol l⁻¹ barium is added to normal Ringer’s (containing 2 mmol l⁻¹ calcium) a prolongation of the impulse is also seen, but trains of impulses are not. The ability of barium to prolong the action potential as well as substituting for calcium as a current carrier has been observed in many preparations, and has been related to a blocking action on potassium channels (Sperelakis, Schneider & Harris, 1967).

Given that calcium is the current carrier in the salivary cell action potential, it is predicted that the overshoot should vary with external calcium concentration, and that removal of calcium in normal sodium will abolish the impulse. Fig. 8 shows that the overshoot increases when external calcium is increased from 2 to 20 mmol l⁻¹, by an amount which averages 25 mV/log concentration change. Furthermore, no active response can be elicited in zero-calcium solution. Studying the effect of reduced calcium is hampered by the tendency of the proboscis musculature to then undergo spontaneous contractions and dislodge the electrodes.

D-600 (methoxyverapamil) acts as a calcium channel antagonist in a variety of tissues. When 2 × 10⁻⁶ mol l⁻¹ D-600 (Gallopamil-HCl, Knoll AG) is washed into the bath, a progressive prolongation of the action potential is seen, (Fig. 9A), although the time course and peak value of the upstroke are unchanged, as is the resting potential. Five minutes after wash-in, a depolarizing pulse no longer evokes an overshooting response, nor is anode-break excitation effective. However, several seconds of large hyperpolarizing current followed by the same depolarizing current evokes a single impulse with a prolonged repolarizing phase. The lower concentration of 5 × 10⁻⁷mol l⁻¹ D-600 also prolongs, then abolishes, the impulse in response to depolarizing pulses, but anode-break excitation now results in a prolonged impulse (Fig. 9B). The phases of repolarization are accentuated, and there is an obvious ‘rebound’ after the initial fast phase. An interaction between membrane potential, action potential frequency and the Ca²⁺ current blocking properties of the organic calcium antagonists is well documented (Triggle, 1981). Our data suggest that the antagonism may be partially relieved by hyperpolarization of the membrane : we have not studied frequency dependence of D-600 effects.

External application of 10 mmol l⁻¹ tetraethylammonium (TEA) reversibly prolongs the action potential (Fig. 10). The slower phase of repolarization is delayed, and impulses of 8–10 s duration are seen. TEA always inhibits calcium-induced potassium fluxes when applied at the inner membrane surface, but also in some cases (skeletal muscle, molluscan neurones) when applied to the outer surface (Schwarz & Passow, 1983). TEA is also known to block voltage-sensitive potassium channels, and so this prolongation of the impulse does not allow us to draw a firm conclusion about the type of K⁺ channels present. The pronounced after hyperpolarization is markedly reduced in the presence of TEA, suggesting that this undershoot is due to elevated potassium permeability.

Exocrine tissues in a wide range of species are composed of cells which are in cytoplasmic continuity via gap junctions, detected by electrical or dye coupling between adjacent groups of cells (Petersen, 1980). Many exocrine glands are inexcitable, in that they do not produce any kind of action potential, and these are exemplified by mammalian salivary and lacrimal glands, exocrine pancreas and insect salivary glands (Petersen, 1976; House, 1980). However, electrical excitability has been demonstrated in mammalian endocrine gland cells, and in the salivary glands of nine genera of gastropod molluscs, whose action potentials propagate from cell to cell along the glandular epithelium (Kater, Rued & Murphy, 1978). Like previously studied exocrine glands, these molluscan salivary glands are composed of cells which are electrically inter-coupled via low-resistance junctions.

The leech salivary gland described in this report is novel, for it is composed of Unicellular, exocrine gland cells which are excitable. The tissue is properly termed a salivary gland ; cells release their contents into the anterior portion of the digestive tract, and electrophoretic analysis has shown that gland protein extracts contain anticoagulant and fibrinolytic activity, which is correlated with the animal’s blood diet (Budzynski et al. 1981a; Budzynski, Olexa & Sawyer, 1981b). The cells cannot be considered neurosecretory, for their site of release is not associated with either neural or haemal elements.

The cortical arrangement of the salivary cell bodies and medullary location of the ductules has led Sawyer et al. (1982) to comment that the anterior gland is ‘closely reminiscent of a (leech) central nervous system’. Despite our finding of electrical excitability, this seems not to be a useful analogy, for the cells are clearly not neuronal, and the medullary processes are not part of an integrative neuropile, but are totally independent of one another. On the basis of histology and histochemistry, they concluded that the anterior gland was composed of sub-populations of both serous and mucous cells. They do not indicate whether there is any stereotyped arrangement of the cell bodies of a particular class when the whole gland is viewed. Our studies have not revealed any sub-populations on the basis of electrophysiology.

The importance of calcium in secretion is by now well known (Douglas, 1968, 1974; Rubin, 1974, 1982), and it is generally agreed that an increase in cytosolic calcium ion concentration leads to secretion by exocytosis. Similarly, calcium has been documented as a current carrier in excitable cells (Reuter, 1973; Hagiwara & Byerly, 1981 ; Tsien, 1983), and so it is not surprising to find a calcium component to the action potential of a secretory cell. Several properties of leech giant salivary cells make them unusually amenable to electrophysiological studies, and suggest that the preparation will prove useful for investigation of both the properties of ion channels and the process of secretion.

Firstly, the salivary cells are exceptionally large, many over 200μm in diameter, and the largest exceeding 1 mm in larger animals. Secondly, the cell bodies are relatively stable within the gland, and it is routinely possible to impale them with one or two microelectrodes under visual control. Thirdly, the absence of coupling in leech giant salivary cells enables them to be studied singly in situ. In other glands, where electrical coupling is present, recordings from one cell are affected by the activity of communicating neighbouring cells. This problem led Goldring, Kater & Kater (1983) to develop enzymatic dissociation techniques to study salivary gland cells from the terrestrial slug Ariolimax, the largest of which exceed 100μm. Fourthly, the results show that we have been able to satisfy most of the tests suggested by Hagiwara & Byerly (1981) to identify calcium-dependent action potentials. Namely, the overshoot varies with external calcium, and does not change in the absence of sodium. The impulse is blocked by polyvalent cations, is unaffected by high concentrations of TTX, and persists when barium or strontium substitute for calcium.

The prolongation of the action potential both by procedures which are known to interfere with calcium currents, and by TEA, suggests that there may be a calcium-sensitive potassium conductance mechanism in the salivary cell membrane. Thus, reduced inward calcium current would delay the activation of a repolarizing potassium current. However, prolongation might also result from reduced inactivation of calcium channels whose inactivation is sensitive to intracellular calcium levels. The presence of sodium channels in the membrane has not been excluded, although the low rate of rise of the action potential and lack of change in overshoot when sodium is removed indicate that any inward sodium current is of low density. Successful application of a voltage clamp technique to allow measurement of ionic currents could determine if any sodium currents were detectable. If, as our present results suggest, sodium currents are absent, these cells might provide a preparation for studying both calcium channels and cellular secretion.

This research was supported in part by NIH Grant NS 14492 (CML).