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5-Hydroxytryptamine Stimulates Net Ca²⁺ Flux in the Ventricular Muscle of a Mollusc (Busycon canaliculatum) During Cardioexcitation

Department of Biology, Penn State University, Abington College, Abington, Pennsylvania 19001

E-mail: cld5{at}psu.edu

	Abstract

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Noninvasive, self-referencing calcium (Ca²⁺) electrodes were used to study the mechanisms by which 5-hydroxytryptamine (5-HT) affects net Ca²⁺ flux across the sarcolemma of myocytes from ventricular trabeculae (from a marine gastropod, Busycon canaliculatum). Treatment of isolated trabeculae with 5-HT causes a net Ca²⁺ efflux, which is 30% blocked by verapamil. These findings suggest that the efflux is in part the result of a previous Ca²⁺ influx through L-type Ca²⁺ channels and is due to a rapid Ca²⁺ extrusion mechanism inherent to the sarcolemma of these myocytes. 5-HT-induced net Ca²⁺ efflux is also reduced by about 40% by treatment with a sodium (Na⁺)-free, lithium (Li⁺)-substituted saline, which shuts down the Na-Ca exchanger during Ca²⁺ extrusion. Cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum (SR) Ca²⁺ ATPase, almost completely abolishes the 5-HT-induced net Ca²⁺ efflux, suggesting that the SR rather than the extracellular pool is the primary Ca²⁺ reservoir serving 5-HT-induced excitation.

	Introduction

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The rhythmicity of molluscan cardiac muscle is regulated by numerous neurotransmitters and neuromodulatory agents, the most widely studied of these being the biogenic amine 5-hydroxytryptamine (5-HT), the tetrapeptide FMRFamide, and acetylcholine (ACh). Pharmacological application of 5-HT onto the isolated hearts of the gastropods Busycon canaliculatum (Hill, 1958; Huddart and Hill, 1996), Lymnaea stagnalis (Buckett et al., 1990), and Aplysia californica (Liebeswar et al., 1975) causes a dose-dependent increase in inotropic and chronotropic activity with a primary effect on long-duration chronotropic regulation. Underlying this potentiation in cardiac rhythmicity is an increase in the amplitude and frequency of cardiac action potentials as well as the coupled systolic force (Hill et al., 1992). The combined anatomical, pharmacological, and physiological studies on the gastropods Aplysia, Busycon, and Lymnaea have provided overwhelming evidence that 5-HT acts as a cardioexcitatory neurotransmitter in this class of mollusc.

⁴⁵Ca²⁺ efflux studies have been conducted on a variety of molluscan cardiac and smooth muscles to determine how Ca²⁺ ions may be mobilized by 5-HT during the process of contraction or relaxation (Bloomquist and Curtis, 1972, 1975; Koch and Greenberg, 1981; Sawada et al., 1984; Ishii et al., 1989). The present study uses a newer technique to study Ca²⁺ flux, noninvasive self-referencing Ca²⁺ electrodes, to determine possible mechanisms by which 5-HT affects trans-sarcolemmal net Ca²⁺ flux in the Busycon ventricle as a clue to understanding its role during the excitation-contraction (E-C) coupling in the gastropod heart. An earlier study focused on the action of FMRFamide on net Ca²⁺ flux in the ventricle of Busycon canaliculatum (Devlin, 1997), so we may be able to compare mechanisms working at putative Ca²⁺ pools accessed by 5-HT and FMRFamide to achieve the same end—enhanced cardiac performance.

	Materials and Methods

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Specimens of Busycon canaliculatum, the channeled whelk, were obtained on the day of each experiment from the Marine Resources Center of the Marine Biological Laboratory (MBL), Woods Hole, Massachusetts. All experiments were conducted at the National Institutes of Health BioCurrents Research Center located at the MBL.

Preparation
The shell was completely cut away from the animal with bone forceps, exposing the heart and its enveloping opaque pericardium. The pericardium was opened, and the ventricle was removed by severing its connections to the aorta and atrium. The ventricle was opened and pinned down in a dish of natural seawater to expose the inner latticework of trabeculae. An individual trabecula was isolated with fine scissors from the inner wall of the ventricle and secured with minuten pins to a Sylgard well in a small recording chamber. The trabecula was bathed in a nominally Ca²⁺-free artificial seawater (ASW) prepared according to the MBL formula (423 mM Na⁺, 9.7 mM K⁺, 9.9 mM Ca²⁺, 51.2 mM Mg²⁺, 538.6 mM Cl^-, 27 mM SO₄^-2, 2.3 mM HCO^3-). Magnesium was used to replace the 9.9 mM Ca²⁺ omitted from the Ca²⁺ -free ASW. The nominal (or background) amounts of Ca²⁺ ions still present in the Ca²⁺-free ASW were typically 100 micromolar (µM) or less and were monitored continuously with a Ca²⁺ electrode throughout the experiment.

Ion flux measurement
The self-referencing ion electrode technique is used to detect net ion flux generated from a biological source, in this case, the sarcolemma of myocytes from a Busycon trabecula. The net ion flux across the membrane is the sum of both inward and outward ion movements. In the present experiments, electrodes loaded with a Ca²⁺-specific ionophore (Fluka Chemika Ca²⁺ ionophore—cocktail A with the neutral carrier ETH 1001) were used to detect net Ca²⁺ flux (in pmol cm^-2 s^-1) across the sarcolemma of the myocytes. The electrode was programmed with PC-based software (Ionprobe) to oscillate with an excursion of 10 µm and a slow frequency of 0.3 Hz; this minimized mixing of the bathing saline. The difference in voltage (µV) between the two ends of the excursion was measured and could be taken as corresponding to a Ca²⁺ concentration gradient, since the excursion was constant.

To construct the electrodes, borosilicate micropipettes were pulled from 1.5-mm-diameter glass capillaries (World Precision Instruments, Inc.), then back-filled with 100 mM CaCl₂ in 0.1% agar. The pipette was front-filled with calcium ionophore—cocktail A containing neutral carrier ETH 1001 (Fluka Chemika) to produce an ionophore column of 40 µm. A Ag/AgCl wire inserted into the back of the micropipette served as the coupling to the headstage. To calibrate the electrode prior to the experiment, the electrode was tested in 0.1 mM, 1 mM, and 10 mM Ca²⁺ solutions to check its Nernstian properties—that is, an approximate 28 mV difference per decade change in Ca²⁺ concentration. The return electrode was a Ag/AgCl wire inserted into a glass capillary containing 3 M KCl agar.

The electrode oscillated at right angles to the long axis of the trabecula and was positioned at two distances from the muscle surface. One electrode position was 500 µm away from the muscle surface. At this relatively great distance, Ca²⁺ flux from the ion source (i.e., the muscle) cannot be detected. Thus, only the control, background levels of Ca²⁺ in the saline are recorded at this position. The second electrode position was only 5 µm from the muscle surface, so net Ca²⁺ flux could be measured directly at the sarcolemma. From the background level of Ca²⁺ ions in the bathing saline, the Ca²⁺ ion concentration gradient at the muscle surface, and the diffusion constant for Ca²⁺ ions, net Ca²⁺ ion flux was calculated using a modification of the Fick equation. The mathematical formulas used in the conversion of voltage to flux units, and other technical aspects and applications of the self-referencing electrode technique, are described by Smith et al. (1999).

Pharmacological agents
The neurotransmitter, 5-HT, and the L-type channel blocker, verapamil, were obtained from Sigma Chemical Company (St. Louis, MO). Verapamil was selected over other Ca²⁺ blockers because of its consistent antagonistic action on other invertebrate cardiac and smooth muscle types (Devlin 1993a, b, 1997, Devlin and Smith, 1996). Diltiazem or nifedipine, which often act as agonists in molluscan muscle preparations, were not used in this study. The Ca²⁺ channel agonist, Bay K 8644, and the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) inhibitor, cyclopiazonic acid (CPA), were obtained from Research Biochemicals International (Natick, MA). At the beginning of each experiment each drug was diluted in the nominal Ca²⁺-free ASW to achieve the designated experimental concentration.

Experimental protocol
The following general protocol was used throughout the present experiments. An initial net Ca²⁺ efflux generated by 10^-7 M 5-HT (the control) was recorded during a 3-min treatment; this net Ca²⁺ efflux immediately returns to basal levels when the 5-HT is washed from the trabecula by flushing with ASW for 20 to 30 min. Next, the trabecula was pretreated for 10 min with verapamil or Bay K 8644 (10^-5 M) or other inhibitors (CPA or Li⁺ ASW). 5-HT (10^-7 M) dissolved in solutions of verapamil or Bay K 8644 (10^-5 M) or other inhibitors (CPA or Li⁺ ASW) was reapplied to the trabecula for a 3-min treatment period. The control, the initial response to 5-HT alone, was then compared to the response to 5-HT in the presence of the agonist, antagonist, or inhibitor. A t-test analysis was then performed.

	Results

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With the electrode placed 5 µm from the muscle surface, a basal net Ca²⁺ efflux of 1.21 ± 0.42 pmol cm^-2 s^-1 (n = 10) is recorded from the trabecula at rest in Ca²⁺-free ASW. A range of 5-HT concentrations (10^-12 M to 10^-5 M) was then tested. Above a threshold of about 10^-10 M, all concentrations of 5-HT enhance basal net Ca²⁺ efflux. The maximal efflux is induced at 5-HT between 10^-8–10^-7 M; above 10^-7 M, the net Ca²⁺ efflux is actually smaller than those induced by lower 5-HT concentrations (Fig. 1). Thus, the 5-HT receptor was desensitized by exposures to higher doses of its ligand. Because the effect of 10^-7 M 5-HT does not desensitize the receptor and is completely reversible, it was chosen as the concentration to be challenged by various Ca²⁺ channel antagonists or agonists. A stable net Ca²⁺ efflux induced by 10^-7 M 5-HT is on the order of 2.63 ± 1.01 pmol cm^-2 s^-1 (n = 8).

View larger version (20K): [in this window] [in a new window]   Figure 1. An experimental record showing a concentration-dependent effect of 5-HT on net Ca2+ efflux (in pmol cm-2 s-1). ASW = spontaneous net Ca2+ flux measurement taken when the electrode was placed directly at the ion source, the trabecula, at a distance of less than 5 µm during treatment with an artificial Ca2+-free seawater (ASW). 5-HT (10-10 M to 10-5 M) = net Ca2+ efflux measurement taken when the electrode was still placed directly at the trabecula at a distance of less than 5 µm during treatment with a range of 5-HT concentrations. Maximum net efflux was recorded between 10-8 M and 10-7 M 5-HT. Background (BG) = small oscillations around the baseline that were recorded when the electrode was placed 500 µm from the ion source, the trabecula.

View larger version (19K): [in this window] [in a new window]   Figure 2. An experimental record showing that the 5-HT-induced net Ca2+ efflux signal (in pmol cm-2 s-1) is inhibited by the L-type channel antagonist verapamil (10-5 M). ASW = spontaneous net Ca2+ efflux measurement taken when the electrode was placed directly at the ion source, the trabecula, at a distance of less than 5 µm. This signal was recorded prior to any drug treatment when the trabecula was bathed only in an artificial Ca2+-free seawater (ASW). 5-HT (1st) = net Ca2+ efflux measurement taken when the electrode was placed directly at the trabecula during treatment with 10-7 M 5-HT. Verapamil = net Ca2+ efflux measurement taken when the electrode was placed directly at the trabecula after a 10-min treatment with verapamil (10-5 M). 5-HT in ver = net Ca2+ efflux measurement taken when the electrode was placed directly at the trabecula when treated with 5-HT (10-7 M) in verapamil (10-5 M). After verapamil was washed from the muscle, the final response to 5-HT (2nd) was greatly enhanced. All above recordings at the trabecula were taken at distance of 5 µm or less. Background (BG) = small oscillations around the baseline that were recorded when the electrode was placed 500 µm from the ion source, the trabecula.

View larger version (17K): [in this window] [in a new window]   Figure 3. The inhibitory effect of LiASW on 5-HT-induced net Ca2+ efflux (in pmol cm-2 s-1). First bar, background (BG): Net Ca2+ efflux measurement taken when the electrode was placed 500 µm from the ion source, the trabecula. Second bar, spontaneous flux: Net Ca2+ efflux measurement taken when the electrode was placed directly at the ion source, the trabecula, at a distance of less than 5 µm during treatment with an artificial seawater. Third bar, 5-HT control: Net Ca2+ efflux measurement taken when the electrode was still placed directly at the trabecula during treatment with 5-HT (10-7 M). Fourth bar: After washing the muscle for 30 min with artificial seawater, net Ca2+ efflux measurement was taken when the electrode was placed directly at the ion source, the trabecula, at a distance of less than 5 µm during treatment with a Na-free, Li-substituted artificial seawater (LiASW). Fifth bar, 5-HT in LiASW: Net Ca2+ efflux measurement taken when the electrode was still placed directly at the muscle during treatment with 5-HT (10-7 M) in LiASW. The data are means pooled from three muscles ± SD.

View larger version (15K): [in this window] [in a new window]   Figure 4. The inhibitory effect of SERCA inhibitor, cyclopiazonic acid (CPA) on 5-HT-induced net Ca2+ efflux (in pmol cm-2 s-1). First bar, spontaneous flux: Net Ca2+ efflux measurement taken when electrode was placed directly at the ion source, the trabecula, at a distance of less than 5 µm during treatment with an artificial seawater. Second bar, 5-HT control: Net Ca2+ efflux measurement taken when the electrode was still placed directly at the trabecula during treatment with 5-HT (10-7 M). Third bar, 5-HT in CPA: Net Ca2+ efflux measurement taken when the electrode was still placed directly at the muscle during treatment with 5-HT (10-7 M) in CPA (10-5 M). The data are means pooled from three experiments on three ventricles ± SD.

	Discussion

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Verapamil, an L-type Ca²⁺ channel blocker, inhibits 5-HT-induced net Ca²⁺ efflux by about 30% in myocytes of the Busycon trabeculae. Similarly, in both gastropod (Huddart and Hill, 1996) and bivalve ventricles (Devlin, 1993)—where 5-HT enhances both inotropic and chronotropic responses—the effect of 5-HT is dependent on the movement of extracellular Ca²⁺ ions through an L-type channel, since 5-HT responses are blocked by verapamil (Devlin, 1993b). Although verapamil has no significant effect on basal net Ca²⁺ efflux in the Busycon ventricle, it does inhibit the 5-HT response, suggesting a use-dependent mechanism of block. This same use-dependent block by verapamil also occurs in many other invertebrate and vertebrate muscle preparations that were either chemically or electrically stimulated (Lee and Tsien, 1983; Zahradnik and Zachar, 1983; Vaghy et al., 1988; Nanasi et al., 1990).

Whereas verapamil inhibits the 5-HT-induced net Ca²⁺ efflux from the Busycon cardiac myocytes by about 30%, it reduces the FMRFamide response by 60% in the same preparation (Devlin, 1997). Therefore, 5-HT relies less on extracellular Ca²⁺ ions than does FMRFamide during the process of E-C coupling in the Busycon ventricle. The results also point to two distinct Ca²⁺ release mechanisms used by these respective neurotransmitters. However, both 5-HT and FMRFamide activate L-type Ca²⁺ channels (whether directly or indirectly remains to be determined) during cardioexcitation, since verapamil partially inhibits net Ca²⁺ efflux induced by either chemical. This finding also indicates that the net Ca²⁺ efflux is in part an effect of an earlier Ca²⁺ influx through L-type channels, possibly carried by the HVA (high-voltage activated) current described by Yeoman et al. (1999).

5-HT (from 10^-9 to 10^-6 M) depolarizes the heart cells from Helix pomatia (Kiss and S.-Rosza, 1978), Aplysia dactylomela (Sawada et al., 1984), and Dolabella auricularia (Hill, 1974). This is the same concentration range in which 5-HT stimulates a net Ca²⁺ efflux from the ventricular myocytes of B. canaliculatum. Two depolarizing Ca²⁺ currents, designated the LVA (low-voltage activated), a T-type current, and the HVA (high-voltage activated), an L-type current, have been identified in the ventricular cells of the gastropod Lymnaea (Yeoman et al., 1999). The sequential activation of the LVA and HVA currents provides the mechanism for pacemaking, AP generation, and a Ca²⁺ source for E-C coupling in the gastropod ventricle (Yeoman et al., 1999). Since 5-HT increases the Ca²⁺-dependent (and a Na⁺-dependent) component of cardiac APs and coupled systolic force in bivalve ventricles (Devlin, 1993b), potentiation of an HVA-like current may be involved given that the Ca²⁺-dependent component is sensitive to verapamil, diltiazem, and Bay K 8644. 5-HT also induces a Ca²⁺ current in Aplysia RB neurons (Pellmar, 1984) and mammalian neurons (Burnashev, 1998).

The relationship between the gastropod 5-HT receptor and coupled ion channels (such as the L-type channel) remains unclear because the subtypes that mediate excitatory responses in the gastropod heart are still being pharmacologically identified. However, a large body of evidence suggests that the 5-HT receptor from both gastropod (S.-Rozsa and Kiss, 1976; Kebabian et al., 1979; Mandelbaum et al., 1979; S.-Rozsa, 1984; Sawada et al., 1984; Drummond et al., 1985; Huddart and Hill, 1996) and bivalve hearts (Higgins, 1974, 1977; Higgins and Greenberg, 1974; Paciotti and Higgins, 1985) is associated with the adenylate cyclase-cAMP signaling pathway, which when stimulated produces an intracellular rise in cAMP. Higgins and Greenberg (1974) found that cAMP increases the phosphorylation of SR proteins that mediate Ca²⁺ sequestration into microsomes prepared from bivalve hearts. This then decreases the length of the diastolic phase of the cardiac cycle and primes the SR to release more Ca²⁺ ions during the next contraction. cAMP was reported to stimulate Ca²⁺ release from intracellular stores in molluscan neurons as well (Kononecko et al., 1983). In contrast, FMRFamide does not appear to work through the adenylate cyclase-cAMP signaling pathway in gastropod ventricles (Drummond et al., 1985; Huddart and Hill, 1996) but instead may mediate phosphoinositide hydrolysis, the process that has been implicated in some bivalve molluscs (Bayakly and Deaton, 1992).

Both 5-HT and some cAMP analogs stimulate ⁴⁵Ca²⁺ efflux from Aplysia ventricular myocytes during enhanced chronotropic and inotropic activities (Sawada et al., 1984). In the present experiments, 5-HT also induces a large net Ca²⁺ efflux (on the order of 2.6 pmol cm^-2 s^-1) from Busycon trabeculae that is recorded even when slow contractions of the trabecula are visible under the microscope. This net Ca²⁺ efflux is sustained over many hours. Sawada et al. (1984) also reported that excitatory drugs that stimulate contraction and cause simultaneous ⁴⁵Ca²⁺ efflux do not deplete cytoplasmic Ca²⁺ in the ventricular myocytes of Aplysia. These combined Ca²⁺ efflux data from Busycon and Aplysia ventricles suggest a substantial internal Ca²⁺ reserve such as the well-developed SR in the gastropod cardiac muscle described by Sanger (1979).

The 5-HT-induced Ca²⁺ efflux recorded from the Busycon ventricle is a large, stable signal that immediately returns to control levels upon washing with seawater. In contrast, in bivalve ventricles (from mussel, Geukensia demissa) that are excited by 5-HT, a transient ⁴⁵Ca²⁺ efflux corresponds to the onset of a 5-HT-induced contracture but is not sustained throughout the contracture (Koch and Greenberg, 1981). When 5-HT is washed from the Geukensia ventricle, a second large ⁴⁵Ca²⁺ efflux occurs, during which total tissue Ca²⁺ increases. Sawada et al. (1984) similarly noted that Ca²⁺ efflux does not necessarily reflect a reduction in cytoplasmic Ca²⁺, because internal reserves may be available. A second application of 5-HT to the mussel heart does not induce a subsequent ⁴⁵Ca²⁺ efflux (Koch and Greenberg, 1981), whereas repeated efflux signals are stimulated by 5-HT in the gastropod heart. These findings reflect the large difference in complexity between the gastropod SR and the bivalve SR as sustainable Ca²⁺ pools.

The SERCA inhibitor, CPA, blocks 5-HT responses by 80% in the Busycon heart, suggesting that the SR is the major Ca²⁺ reservoir used during cardioexcitation by 5-HT. CPA acts by inhibiting the binding of Ca²⁺ ions at high-affinity binding sites on the SERCA; this shuts down both Ca²⁺ uptake and subsequent Ca²⁺ release at the SR of skeletal, cardiac (Balke et al., 1994), and smooth muscle (Suzuki et al., 1992). An indirect effect of inhibiting Ca²⁺sequesteration at the SR is a reduction in Ca²⁺-induced Ca²⁺ release; such a reduction is typically activated by the entry of extracellular Ca²⁺ through voltage-gated Ca²⁺ channels. In short, this would serve to limit the Ca²⁺ needed to activate the contractile proteins. On the other hand, a reduction in Ca²⁺-induced Ca²⁺ release also decreases the Ca²⁺-dependent K⁺ current (I_K-Ca), thus ultimately prolonging the excitability of vertebrate smooth muscle (Suzuki et al., 1992). How CPA is affecting excitability in the mollusc ventricle warrants further investigation, especially considering its effectiveness in blocking Ca²⁺ efflux and the complete reversibility of its action.

The net Ca²⁺ efflux that was recorded during 5-HT treatment was composed primarily of Ca²⁺ released from the SR and secondarily of Ca²⁺, from the extracellular fluid, that had previously entered the myocytes via an L-type current. This net Ca²⁺ efflux reflects the ability of the sarcolemma to rapidly redistribute Ca²⁺ ions outward, therefore preventing the toxic effects of Ca²⁺ overload, and reestablishing intracellular and extracellular Ca²⁺ gradients before the next successive depolarization and contraction. To determine if the net efflux was in part due to activity of the Na⁺-Ca²⁺ exchanger, a Na⁺-free, Li⁺-substituted saline (Li⁺ ASW) was used. The Li⁺ ASW reduces the basal net Ca²⁺ efflux during normal, autorhythmic activity of the Busycon trabeculae by only 20%–30%, whereas the 5-HT and FMRFamide responses are inhibited during Li⁺ treatment by 40% and 73%, respectively (Devlin, 1997). This difference in response suggests a greater reliance on the Na⁺-Ca²⁺ exchanger during chemically mediated excitation than during spontaneous myogenic activity. These Li⁺ substitution experiments on the gastropod trabeculae also show that Na⁺ ions are a necessary stimulus for Ca²⁺ mobilization through voltage-gate channels or from an intracellular pool, perhaps during Na⁺-induced Ca²⁺ release (Lipp and Niggli, 1994), and that they are also involved in the process of Ca²⁺ extrusion. The data from the present study are in agreement with studies on the Na⁺-Ca²⁺ exchanger in other myocytes where the removal of extracellular Na⁺ reduces Ca²⁺ efflux in guinea atrial cells (Reuter and Seitz, 1968) and in internally dialyzed myocytes (Miura and Kimura, 1989).

	Acknowledgments

 
This work was supported by the Marine Biological Laboratory, M. G. F. Fuortes and Lucy B. Lemann Fellowships, the NASA Life Sciences Program, and a Pennsylvania State University Faculty Development Grant. Thanks are also extended to the staff of the N. I. H. BioCurrents Research Center at the MBL for their support in this research.

	Footnotes

 
Received 18 January 2000; accepted 17 February 2001.

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