Regulation of calcium influx into buccal muscles of Aplysia by acetylcholine and serotonin

Acetylcholine (ACh) causes contraction of Aplysia buccal muscles E1 and I5, and serotonin (5-hydroxytryptamine, 5-HT) enhances ACh-elicited contractions of these muscles. Possible roles of calcium influx in mediating these responses were examined by studying influx of 45Ca++. 5-HT increased calcium influx into both I5 and E1. Maximal influx occurred at 10(-6) M 5-HT and the increased influx could be sustained in the presence of 5-HT for at least 10 min. ACh also caused calcium influx, and calcium influx increased approximately in proportion to log[ACh] from 10(-5) M to 10(-3) M ACh. 5-HT and ACh probably bring about calcium influx by different mechanisms since the effect of ACh was additive to a maximal 5-HT response, and 10(-4) M hexamethonium bromide inhibited the increased influx caused by ACh but did not affect influx caused by 5-HT. Cyclic AMP analogues and forskolin neither caused an increase in calcium influx nor an increase in the influx caused by ACh. The data support a model in which ACh-elicited contractions of I5 and E1 are due primarily to calcium entry across the extracellular membrane, and 5-HT can "load" an intracellular site by a mechanism different from that activated by ACh. The data do not support a role for cyclic AMP in mediating the calcium influx response to 5-HT.     

Amiloride inhibits contraction and serotonin modulation of Aplysia muscle

1. Serotonin (5-HT) potentiates acetylcholine (ACh)-elicited contractions of Aplysia buccal muscles. Serotonin potentiation was significantly reduced by 0.03 mM, 0.1 mM, and 0.3 mM amiloride. 2. Unpotentiated ACh-elicited contractions were significantly reduced by 0.1 mM and 0.3 mM amiloride. 3. Amiloride reduced ACh-elicited depolarization. The reduction in contraction caused by 0.3 mM amiloride (to 16% of control) was larger than could be explained by the reduction in depolarization (86% of control). 4. Amiloride had no effect on tension in skinned muscle fibers, indicating that amiloride probably did not have a direct effect on contractile mechanisms. 5. Potentiation of contraction produced by zero sodium (Tris substituted, 0 Na-Tris) medium could be abolished by 0.3 mM amiloride. 6. Zero Na-Tris increased 45Ca influx 2.7-fold. In the presence of 0.3 mM amiloride, 0 Na-Tris increased 45Ca influx only 1.4-fold. 7. Amiloride (0.3 mM) reduced the elevation of muscle cAMP caused by 10(-6) M 5-HT by 60%. Zero Na-Tris did not cause a change in muscle cAMP.     

Voltage-dependent calcium current in dissociated smooth muscle cells of the buccal mass of Aplysia

Summary Isolated smooth muscle cells of the buccal mass of Aplysia contracted in response to depolarization elicited by a patch electrode in whole-cell configuration. With cesium-containing pipet solution and tetraethylammonium and 4-aminopyridine in the external solution depolarization elicited inward current. The voltage-dependent inward current was blocked completely by lanthanum (10 mmol·1^-1), inhibited 80–90% by nifedipine (1 mol·l^-1), and was dependent upon extracellular calcium. These results showed that the voltage-dependent inward current was due to activation of voltage-dependent calcium channels (VDCaCH). Minimal depolarization to begin activating VDCaCH was-60 to-30 mV. Inward current peaked within 8 ms and then decreased rapidly to a lower level of relatively non-inactivating current. The initial peak current could be mostly inactivated by a depolarization to-20 mV for 500 ms. Nifedipine reduced both the peak current and the relatively non-inactivating current. Nifedipine inhibited high potassium-elicited contractions of both intact and dissociated muscle. These results suggested that VDCaCH mediates calcium influx which triggers contraction in molluscan smooth muscle fibers.Abbreviations ACh acetylcholine - ATP adenosine triphosphate - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid - GTP guanosine triphosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - MCG metacerebral giant cell - RNI relatively non-inactivating - SCP small cardioactive peptide - TEA-4AP-IO external solution containing Instant Ocean, tetraethylammonium chloride, and 4-aminopyridine (described in Methods) - TEA tetraethylammonium chloride - VDCaCH voltagedependent calcium channel - 4-AP 4-aminopyridine - 5-HT 5-hydroxytryptamine or serotonin     

Serotonin-activated adenylate cyclase and the possible role of cyclic AMP in modulation of buccal muscle contraction in Aplysia

The possible role of cyclic AMP in mediating opposite modulatory effects of serotonin (5-HT) on Aplysia buccal mass muscles E1 and E2 was examined. Serotonin enhances E1 contractions and inhibits E2 contractions. Adenylate cyclase in membranes of both E1 and E2 is stimulated approximately 180% by 10(-6) M 5-HT and 300% by 10(-3) M 5-HT. Dibutyryl cyclic AMP and 8-benzylthio cyclic AMP mimicked the effect of 5-HT on E1 but had no effect on E2. Theophylline (Th) and isobutylmethylxanthine (IBMX) mimicked the effect of 5-HT on E1 at high concentrations. Concentrations of Th and IBMX low enough not to have any direct effect on contraction increased both the magnitude and duration of the effect of 5-HT on E1 contraction. Neither Th nor IBMX had a direct effect on E2 contraction, although Th produced a small increase in the effect of 5-HT on E2. These data are consistent with the hypothesis that cyclic AMP mediates the enhancement effect of 5-HT on E1 contraction. Other mechanisms probably mediate the effect of 5-HT on E2 contraction.     

Antagonists of cholinergic and serotonergic responses of Aplysia buccal muscle

Cholinergic and serotonergic receptors of Aplysia californica buccal muscles were characterized pharmacologically by determining compounds that effectively inhibited contractile responses to acetylcholine (ACh) and modulatory effects of serotonin (5-HT), respectively. pA50 for ACh to elicit contraction averaged 4.7 ± 0.1 (mean ± SE, equivalent to 2 × 10⁻⁵ M). Both hexamethonium bromide and atropine inhibited ACh-elicited contractions, but neither inhibited the response completely, nor were the two together able to antagonize the response completely. Curare caused inhibition only at low ACh doses, and muscarinic antagonists pirenzapine and 4-diphenylacetoxy-N-methylpiperidine methiodide caused partial inhibition. The most effective blocker of ACh-elicited contractions was the nicotinic antagonist mecamylamine. 10⁻⁴M mecamylamine completely blocked the cholinergic response. ACh contractions were inhibited 90% within 10 min and took >40 min to recover from mecamylamine. Specificity was indicated by the lack of effect of mecamylamine on potassium-elicited contraction. NAN-190 blocked the potentiating effect of 5-HT without having inhibitory or potentiating effects by itself on ACh-elicited contractions. NAN-190 blocked the potentiating effect of 8-OH-DPAT. Cholinergic receptors on Aplysia buccal muscles are most effectively inhibited by mecamylamine and may have mixed nicotinic/muscarinic character. Serotonergic receptors have pharmacological similarities to vertebrate 5-HT_1A receptors and may be closely related to the gastropod 5-HTlym receptor.     

Block of contracture in skinned frog skeletal muscle fibers by calcium antagonists

The ability of a number of calcium antagonistic drugs including nitrendipine, D600, and D890 to block contractures in single skinned (sarcolemma removed) muscle fibers of the frog Rana pipiens has been characterized. Contractures were initiated by ionic substitution, which is thought to depolarize resealed transverse tubules in this preparation. Depolarization of the transverse tubules is the physiological trigger for the release of calcium ion from the sarcoplasmic reticulum and thus of contractile protein activation. Since the transverse tubular membrane potential cannot be measured in this preparation, tension development is used as a measure of activation. Once stimulated, fibers become inactivated and do not respond to a second stimulus unless allowed to recover or reprime (Fill and Best, 1988). Fibers exposed to calcium antagonists while fully inactivated do not recover from inactivation (became blocked or paralyzed). The extent of drug-induced block was quantified by comparing the height of individual contractures. Reprimed fibers were significantly less sensitive to block by both nitrendipine (10 degrees C) and D600 (10 and 22 degrees C) than were inactivated fibers. Addition of D600 to fibers recovering from inactivation stopped further recovery, confirming preferential interaction of the drug with the inactivated state. A concerted model that assumed coupled transitions of independent drug-binding sites from the reprimed to the inactivated state adequately described the data obtained from reprimed fibers. Photoreversal of drug action left fibers inactivated even though the drug was initially added to fibers in the reprimed state. This result is consistent with the prediction from the model. The estimated KI for D600 (at 10 degrees and 22 degrees C) and for D890 (at 10 degrees C) was approximately 10 microM. The estimated KI for nitrendipine paralysis of inactivated fibers at 10 degrees C was 16 nM. The sensitivity of reprimed fibers to paralysis by D600 and D890 was similar. However, inactivated fibers were significantly less sensitive to the membrane-impermeant derivative (D890) than to the permeant species (D600), which suggests a change in the drug-binding site or its environment during the inactivation process. The enantomeric dihydropyridines (+) and (-) 202-791, reported to be calcium channel agonists and antagonists, respectively, both caused paralysis, which suggests that blockade of a transverse tubular membrane calcium flux is not the mechanism responsible for antagonist-induced paralysis. The data support a model of excitation-contraction coupling involving transverse tubular proteins that bind calcium antagonists.     

Excitatory effects of cholinergic,adrenergic and glutaminergic agonists on a buccal muscle of Aplysia

The anterior extrinsic protractors in the buccal mass of Aplysia are symmetrical sheets of branching bundles of muscle fibers which form an electrical syncytium. The addition of potassium or cholinergic, adrenergic, and glutaminergic agonists to the sea water bathing medium produces contracture of the muscle. Strychnine and cholinergic or adrenergic antagonists all block contractures produced by cholinergic and adrenergic agonists but not those produced by potassium or glutamate. Iontophoretic application through microelectrodes of acetylcholine or dopamine anywhere on the muscle surface produced a graded depolarization of the membrane. By contrast, glutamate produces depolarization only at discrete membrane sites. Endogeneous contractions often appeared spontaneously or could be induced by drug exposure. ATP inhibits endogeneous contractions.     

Enhancement of inward current by serotonin in neurons of Aplysia

In RB cells of Aplysia, serotonin, in the presence of TEA, 4AP and Ba, elicits a voltage-dependent inward current. In Ba-TEA-4AP seawater, RB cells showed a negative slope region (NSR) in their current-voltage (I-V) relationship when measured at the end of 2-s commands from a holding potential of -60 mV. Addition of serotonin to the bathing solution enhanced the NSR. When holding potential was lowered to -10 mV, the NSR as well as the effects of serotonin were greatly reduced. Addition of 20 mM cobalt to the bathing solution blocked both the NSR and the inward current produced by serotonin. Changes in potassium concentration produced no consistent shift in voltage sensitivity nor change in amplitude of the current elicited by serotonin. Intracellular injection of cesium sufficient to broaden action potentials did not block the enhancement of NSR by serotonin. These results support the conclusion that in RB cells, serotonin produces a voltage-dependent current carried by calcium ions.     

Synaptic connections and functional organization in Aplysia buccal ganglia

The buccal ganglion of Aplysia contains three morpho-functional groups (A, B, and C) of large cells and two groups (s₁ and s₂) of small cells. The A cells evoke monosynaptic IPSPs in the B cells. We found that s₁ cells can evoke large EPSPs in the A cells, IEPSPs in the B cells, and EIIPSPs in the C cells; several s₁ cells are able to evoke all three types of responses. Many s₂ cells can evoke these same responses, but only in the A and B cells. Furthermore, the s cells can evoke depolarizing PSPs in other s cells; this relation is often reciprocal. All these responses may also be contralateral. Their monosynaptic nature is shown by the consistent 1:1 relationship with the presynaptic spike, and also by the effects of intracellular tetraethylammonium and of high Mg²⁺ concentration in the bathing medium. D-tubocurarine reversibly suppresses the I phase of the IEPSP evoked by the s cells in the B cells. All the responses evoked by the s cells undergo depression with repetition. The network formed by all these relations is outlined, and a double relationship proposed between s cells and B cells. By electrophysiological tracing of axonal pathways it is shown that the A cells send axons into the 3rd buccal nerve, the B cells into the 2nd and/or 3rd buccal nerve and in two cases into the redular nerve, and the C cells into the gastro-oesophageal nerve. Spontaneous synaptic activity of the buccal neurons appears to be formed mostly by the described PSPs. Spontaneous firing inside the isolated ganglion corresponds well to the alternate pattern of muscular contractions of the buccal mass.     

Involvement of pedal peptide in locomotion in Aplysia: modulation of foot muscle contractions

Pedal peptide (Pep) is a 15-amino-acid neuropeptide that is localized within the Aplysia central nervous system (CNS) predominantly to a broad band of neurons in each pedal ganglion. Pep-neurons were identified by intracellular staining and immunocytology or by radioimmunoassay (RIA) of extracts from identified neurons. RIA reveals that 97% of all Pep-like immunoreactivity (IR-Pep) in pedal nerves is found in the three nerves that innervate the foot. Nearly every Pep-neuron sends an axon out at least one of these three nerves. Application of Pep to foot muscle causes an increase in the amplitude and relaxation rate of contractions driven by nerve stimulation or intracellular stimulation of pedal motor neurons. The increase in relaxation rate was the predominant effect. Intracellular recording in "split-foot" preparations reveals that Pep-neurons increase their overall firing rates and fire in bursts with each step during locomotion. Recovery of IR-Pep from foot perfusate following pedal nerve stimulation increases in a frequency-dependent fashion. Thus it appears that one function of Pep-neurons is to modulate foot muscle contractility during locomotion in Aplysia.     

Extracellular calcium ion depletion in frog cardiac ventricular muscle.

The extracellular free [Ca++] in frog ventricular muscle strips was monitored using single-barrel calcium ion-selective microelectrodes. During trains of repetitive stimulation, a heart rate-dependent, sustained fall (depletion) of the extracellular free [Ca++] occurs, which is most likely a consequence of net Ca++ influx into ventricular cells. The magnitude of the [Ca++]0 depletion increases for higher Ringer's solution [Ca++], and is reversibly blocked by manganese ion. Prolonged repetitive field stimulation (20-30 min) activates additional cellular Ca++ efflux, which can balance the additional Ca++ influx caused by stimulation, resulting in abolition of extratrabecular [Ca++]0 depletion in 20-30 min, and hence zero net transmembrane Ca++ flux at steady state. In the poststimulation period of quiescence, cellular Ca++ efflux persists and causes an elevation (accumulation) of the extracellular free [Ca++]. From these [Ca++]0 depletions, quantitative estimates for the net transmembrane Ca++ flux were derived using an analytical solution to the diffusion equation. In the highest Ringer's solution [Ca++] used (1 mM) the calculated net increase of the total intracellular calcium per beat was 6.5 +/- 1.4 mumol/l of intracellular space. This corresponds to an average net transmembrane Ca++ influx of 0.81 +/- 0.17 pmol/cm2/s during the 800-ms action potential. In lower bath [Ca++] the net transmembrane [Ca++] flux was proportionately reduced.     

Extracellular calcium sensing in rat aortic vascular smooth muscle cells

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.     

Synaptic connections and functional organization in Aplysia buccal ganglia.

The buccal ganglion of Aplysia contains three morpho-functional groups (A, B, and C) of large cells and two groups (s1 and s2) of small cells. The A cells evoke monoxynaptic IPSPs in the B cells. We found that s1 cells can evoke large EPSPs in the A cells, IEPSPs in the B cells, and EIIPSPs in the C cells; several s1 cells are able to evoke all three types of responses. Many s2 cells can evoke these same responses, but only in the A and B cells. Furthermore, the s cells can evoke depolarizing PSPs in other s cells; this relation is often reciprocal. All these responses may also be contralateral. Their monosynaptic nature is shown by the consistent 1:1 relationship with the presynaptic spike, and also by the effects of intracellular tetraethylammonium and of high Mg2+ concentration in the bathing medium. d-tubocurarine reversibly suppresses the I phase of the IEPSP evoked by the s cells in the B cells. All the responses evoked by the s cells undergo depression with repetition. The network formed by all these relations is outlined, and a double relationship proposed between s cells and B cells. By electrophysiological tracing of axonal pathways it is shown that the A cells send axons into the 3rd buccal nerve, the B cells into the 2nd and/or 3rd buccal nerve and in two cases into the radular nerve, and the C cells into the gastro-oesophageal nerve. Spontaneous synaptic activity of the buccal neurons appears to be formed mostly by the described PSPs. Spontaneous firing inside the isolated ganglion corresponds well to the alternate pattern of muscular contractions of the buccal mass.     

Neuron-specific modulation by serotonin of regenerative outgrowth and intracellular calcium within the CNS of Helisoma trivolvis

We have investigated the cell-specific effect of serotonin (5-HT) on regenerating neurons within the adult central nervous system of the pond snail, Helisoma trivolvis. In culture, 5-HT arrests outgrowth of buccal neurons B19 but not neurons B5 (Haydon, McCobb, and Kater, 1984). After axotomy, neurons within the Helisoma nervous system typically exhibit profuse regenerative outgrowth. This study, on neurons within the CNS, shows that 5-HT selectively inhibits the outgrowth of specific identified neurons, and also causes significant elevations in intracellular calcium concentrations as measured by the calcium indicator dye, Fura-2. The outgrowth of neurons B19 and C1 was selectively inhibited when ganglia were incubated in 5 X 10(-5) M 5-HT. The outgrowth of buccal neurons B5, however, was not affected. Moreover, 5-HT caused significant transient elevations of calcium concentrations in neurons B19 over 30 minutes, but neurons B5 did not show any increases in calcium concentrations with the addition of 5-HT. These results suggest that the effect of 5-HT upon outgrowth of regenerating neurons may be due to an increase in the intracellular calcium concentration.     

Biomechanical properties and a kinetic simulation model of the smooth muscle I2 in the buccal mass of Aplysia

The muscle I2 is a smooth muscle from the buccal mass of the marine mollusc Aplysia californica whose neural control, in vivo kinematics, and behavioral role have been extensively analyzed. In this study, we measured the activation and contractile dynamics of the muscle in order to construct a Hill-type kinetic model of the muscle. This is the first study to our knowledge, of Aplysia muscle contractile dynamics. The isometric force-frequency relationship of I2 had a frequency threshold of about 6–8 Hz, and its force output saturated at 20–25 Hz, properties that match the high frequency (20 Hz) bursts generated by the B31/B32 neurons that innervate it. Peak isometric force was generated at about 118% of the in situ relaxed length. These results and I2's estimated in vivo kinematics suggest that it generates maximum force at the onset of protraction. The muscle tension during iso-velocity lengthening and shortening was an asymmetric function of velocity. Short range stiffness and yielding responses were observed in lengthening, whereas muscle tension decreased smoothly in shortening. These visco-elastic properties suggest that the I2 muscle can serve to brake forceful retraction movements. A Hill-type model, parameterized from the measurements, captured many of the mechanical properties of I2. Our results provide a quantitative understanding of the biomechanical significance of the muscle's neural control and provide a basis for simulation studies of the control of feeding behavior. Received: 5 February 1999 / Accepted in revised form: 18 May 1999     

Regulation of protein kinase C Apl II by serotonin receptors in Aplysia

Serotonin (5-hydroxytryptamine, 5HT) is the neurotransmitter that mediates dishabituation in Aplysia. Serotonin mediates this behavioral change through the reversal of synaptic depression in sensory neurons (SNs). However, the 5HT receptors present in SNs and in particular, the receptor important for activation of protein kinase C (PKC) have not been fully identified. Using a recent genome assembly of Aplysia, we identified new receptors from the 5HT(2) , 5HT(4) , and 5HT(7) families. Using RT-PCR from isolated SNs, we found that three 5HT receptors, 5HT(1Apl(a)) , 5HT(2Apl) , and 5HT(7Apl) were expressed in SNs. These receptors were cloned and expressed in a heterologous system. In this system, 5HT(2Apl) could significantly translocate PKC Apl II in response to 5HT and this was blocked by pirenperone, a 5HT(2) receptor antagonist. Surprisingly, pirenperone did not block 5HT-mediated translocation of PKC Apl II in SNs, nor 5HT-mediated reversal of depression. Expression of 5HT(1Apl(a)) in SNs or genistein, an inhibitor of tyrosine kinases inhibited both PKC translocation and reversal of depression. These results suggest a non-canonical mechanism for the translocation of PKC Apl II in SNs.     

Extracellular matrix effect on RhoA signaling modulation in vascular smooth muscle cells

Morphological adaptations of vascular smooth muscle cells (VSMC) to the mechanically active environment in which they reside, are mediated by direct interactions with the extracellular matrix (ECM) which induces physiological changes at the intracellular level. This study aimed to analyze the effects of the ECM on RhoA-induced mechanical signaling that controls actin organization and focal adhesion formation. VSMC were transfected with RhoA constructs (wild type, dominant negative or constitutively active) and plated on different ECM proteins used as substrate (fibronectin, collagen IV, collagen I, and laminin) or poly-l-lysine as control. Morphological changes of the VSMC were detected by fluorescence confocal microscopy and total internal reflection fluorescence (TIRF) microscopy, and were independently verified using adhesion assays and Western blot analysis. Our results showed that the ECM has an important role in cell spreading, adhesion and morphology with a direct effect on modulating RhoA signaling. RhoA activity significantly affected the stress fibers and focal adhesions reorganization, but in a context imposed by the ECM. Thus, RhoA activity modulation in VSMC induced an increased activation of stress fibers and FA formation at 5 h, while a significant inhibition was recorded at 24 h after plating on the different ECM. Our findings provide biophysical evidence that ECM modulates VSMC response to mechanical stimuli inducing intracellular biochemical signaling involved in cellular adaptation to the local microenvironment.     

VPAC receptor modulation of neuroexcitability in intracardiac neurons: dependence on intracellular calcium mobilization and synergistic enhancement by PAC1 receptor activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have been found within mammalian intracardiac ganglia, but the cellular effects of these neuropeptides remain poorly understood. Fluorometric calcium imaging and whole cell patch clamp recordings were used to examine the effects of PACAP and VIP on [Ca2+]i and neuroexcitability, respectively, in intracardiac neurons of neonatal rats. PACAP and VIP evoked rapid increases in [Ca2+]i that exhibited both transient and sustained components. Pharmacological experiments using PAC1 and VPAC receptor-selective antagonists demonstrated that the elevations in [Ca2+]i result from the activation of VPAC receptors. The transient increases in [Ca2+]i were shown to be the product of Ca2+ mobilization from caffeine/ryanodine-sensitive intracellular stores and were not due to inositol 1,4,5-trisphosphate-mediated calcium release. In contrast, the sustained [Ca2+]i elevations were dependent on extracellular Ca2+ and were blocked by the transient receptor channel antagonist, 2-aminoethoxydiphenyl borate, which suggests that they are due to Ca2+ entry via store-operated channels. In addition to elevating [Ca2+]i, both PACAP and VIP depolarized intracardiac neurons, and PACAP was further shown to augment action potential firing in these cells. Depolarization of intracardiac neurons by the neuropeptides was dependent on activation of VPAC receptors and the concomitant increases in [Ca2+]i. Although activation of PAC1 receptors alone had no direct effects on neuroexcitability, PAC1 receptor stimulation potentiated the VPAC receptor-induced depolarizations. Furthermore, enhanced action potential firing was only observed upon concurrent stimulation of PAC1 and VPAC receptors, which indicates that these receptors act synergistically to enhance neuroexcitability in intracardiac neurons.