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Twenty different RFamide neuropeptide analogues were examined for their relative potencies on the ventricles of Busycon canaliculatum and Buccinum undatum and on the atrium of Busycon to determine the essential requirements for activity at the RFamide receptor. None of the neuropeptide studies was inhibitory to natural cardiac rhythmicity or to FMRFamide (Phe-Met-Arg-Phe-NH(2)) or FLRFamide (Phe-Leu-Arg-Phe-NH(2)) responses. Two tripeptides studied were completely without effect, indicating that a minimum of four amino acids in the peptide chain length was essential for any activity. The original parent tetrapeptide FMRFamide was surprisingly less potent than many of the extended chain peptides such as the penta, hepta and decapeptides. These RFamide neuropeptides were strongly inotropic on both ventricles and the atrium, while on the latter they were strongly chronotropic despite several of these peptides being of a non-molluscan origin. Chain length seems to be of little importance for activity at the receptor. Surprisingly, SCP(B) (small cardioactive Peptide B) was not very effective in either Busycon or Buccinum ventricle. What was also clear was that the configuration of the carboxyl terminal was important for activity. Two neuropeptides in this study possessed an Arg-Met carboxyl terminal and were much less effective than FMRFamide, suggesting that an Arg-Phe terminal is most effective in receptor activation.

1. Isolated radular protractor muscles (RPM) of Busycon canaliculatum show a complex response to potassium-free solution. 2. While exposed to a bathing solution lacking K+, the RPM begins to respond with a hyperpolarizing phase. Within minutes, this gives way to a depolarizing phase (DP). After readmission of a bathing solution containing K+, there is an extra hyperpolarizing phase. 3. Acetylcholine over a range of concentrations from 10(-8)M to 10(-3)M progressively enhances the DP. 4. The action of acetylcholine is sodium dependent.

1. The mean resting potential of 22 atrial preparations of Busycon heart was 42.5 mV, examined by the sucrose gap technique. Spontaneous action potentials of 8-18 mV amplitude occurred in repeated cycles of burst activity, generating burst patterned phasic contractile activity. 2. Isolated ventricles showed slow (1-3 beats min-1) constant myogenic contractile activity, suggesting that the primary driving pacemaker may reside in the atrium. 3. The atrial electrocardiogram commenced with a small prepotential leading to a plateau-like phase and terminated with a sharp spike potential. 4. Acetylcholine (ACh) at high concentrations depolarised the atrium by 5-8 mV and induced strong tonic contractures while suppressing spontaneous action potentials, suggesting an overall inhibitory role in downregulating cardiac intrinsic myogenic rhythms. 5. Serotonin (5-hydroxytryptamine, 5HT) was consistently excitatory, enhancing both action potential amplitude and rhythmic contractions by up to 50% at concentrations of 5 x 10(-7) to 10(-5)M. Neither methysergide nor metoclopramide affected atrial responses to 5HT and the 5HT1 antagonist metitipine simply increased action potential discharge in the rhythmic cycle. The vertebrate 5HT1-3 receptor classification is inappropriate to this molluscan preparation. 6. The atrium was very sensitive to the tetrapeptides FMRF- and FLRFamide, but the enhanced phasic contractions were not accompanied by alteration of resting potential or action potential amplitude, suggestive of neuromodulatory upregulation involving a secondary messenger. The related peptide SCP-B was without effect on the preparation, but GAPFLRFamide was excitatory, although much less so than FMRF- and FLRFamide. 7. Neither adenosine and ATP nor guanosine and GTP affected intrinsic atrial electrical or mechanical activity, suggesting that there was no noncholinergic, nonaminergic element to cardiac neuromodulation in this species. Only ACh, 5HT and FMRF/FLRFamide could be assigned clear roles in this respect.

1. Calcium-free media usually caused a cessation of all electrical and mechanical activity of the Busycon atrium. Where any electrical activity survived, the action potential consisted of a pre- and plateau-like potential devoid of the usual terminal spike. 2. High Ca salines induced tonic force, membrane depolarization and reduction in generation of spontaneous action potentials. The Ca ionophore A23187 enhanced contractions and the SR CaATPase inhibitor cyclopiazonic acid induced slight depolarization, tonic contractures and increased action potential firing. 3. The inorganic Ca antagonist Co2+ was without effect on the preparations, although the lanthanide Gd3+ inhibited contractions and spontaneous action potentials as well as inducing membrane potential depolarization. 4. The organic Ca entry-blocker nifedipine enhanced both spontaneous action potential amplitude and the phasic contractions they generated. 5. High K salines considerably depolarized atrial preparations with accompanying large tonic contractures and suppression of action potentials. The K channel-blocker 4AP enhanced action potential amplitude with slight increase in contractions, and TEA depolarized the atrium, and enhanced action potentials and rhythmic contractions. 6. Sodium-free salines strongly hyperpolarized atrial preparations and abolished spontaneous action potentials and, on washout, the membrane potential became temporarily unstable. In 2 preparations, low chloride and chloride-free media induced significant membrane potential hyperpolarization. 7. It is concluded that, in the atrium, the resting membrane potential is largely determined by the transmembrane K gradient, but with significant conductances to Na and Cl though probably not Ca. The action potential spike appears to be a Ca-dependent event and the plateau-like phase may be a Na-dependent event.

1. Isolated cannulated ventricles commenced spontaneous beating on application of perfusion pressure of 10 cm water. Complete hearts showed a fast patterned cyclical rhythm, whereas ventricles devoid of atrial material showed a continuous slow rhythm. 2. Perfused ventricles were inhibited by ACh with a threshold at 10(-8) mol l-1 and arrested at 10(-7) mol l-1, and ventricles under stimulation by 5HT could be arrested by ACh at this concentration. 3. Perfused ventricles were stimulated by 5HT, with threshold at 10(-9) mol l-1 and maximum at 10(-5) mol l-1. Metoclopramide was without affect on 5HT responses, but metitipine and methysergide did inhibit such responses suggesting that the 5HT receptor present possessed mixed properties of the vertebrate 5-HT1 and 5-HT2 receptor subtypes. 4. Ventricles were very sensitive to the excitatory actions of FMRFamide in the 10(-9) to 10(-5) mol l-1 range. Preparations were insensitive to GAPFLRFamide, but SCP-B was modestly excitatory (threshold 10(-7) mol l-1). 5. Preparations were not significantly affected by adenosine, ATP, and guanosine, but GTP was strongly excitatory at 10(-7) mol l-1. 6. 5HT and FMRFamide responses were additive. Preparations responded strongly to the adenylate cyclase activator forskolin and dibutyryl cAMP enhanced spontaneous contractions and 5HT responses, suggesting that the 5HT receptor may operate via a cAMP secondary mechanism. 7. The IP3 inhibitor lithium (10 mmol l-1), caused slight inhibition of FMRFamide responses, suggesting that the receptor to this peptide may operate via IP3 as a second messenger. 8. Neuromodulation in this preparation would appear to involve ACh as inhibitor, 5HT and FMRFamide as upregulators, with no clear roles for FMRFamide-related peptides and GTP.

1. A sucrose gap technique was used to study the effect of a sodium ionophore on the potential changes which occur during superfusion with potassium-free solution. Crucial values were checked with a microelectrode technique. 2. Potassium-free solution induces a complex response consisting of a hyperpolarizing phase (HP) and then a depolarization phase (DP) during exposure to zero K+, followed by a transient extra hyperpolarizing phase (EHP) on readmission of K+. 3. The sodium ionophore, monensin, has the effect of increasing the amplitude of the DP, in perfusion medium of normal Na+ content. This is similar to the effect of treatment with neurohumors and to the after-effect of direct electrical stimulation. The effects of ACh and monensin are additive. 5. These effects are consistent with an action of monensin in increasing Na+ flux in the direction of the concentration gradient and support the hypothesis that neurohumors stimulate the sodium pump by increasing Na+ influx.

Membrane potential and tension of Busycon radular protractor muscles were studied by sucrose gap methods. Excitation-contraction (EC) coupling was examined in response to acetylcholine (ACh) and high K which depolarized the fibres and induced tension, but without action potential firing. Potassium depolarization did not follow predictions expected from the Nernst equation at low and very high K levels, and maximum tension was found at about 100 mM K. EC coupling was very sensitive to [Ca]0. Ca-free media eliminated K- and ACh-induced tension but with normal depolarization, showing full electromechanical uncoupling. Ionophore A23187 enhanced K- and ACh-induced responses and X-537A enhanced ACh responses, demonstrating acute dependence of activation on [Ca]0 in this muscle. The calcium antagonists nifedipine and nisoldipine reduced tension in the muscle only at very high concentrations, and both agents slightly reduced K- and ACh-induced depolarization. Verapamil reduced K- and ACh-induced tension but paradoxically it enhanced the depolarizing actions of these agents leading to electromechanical uncoupling. Abscisic acid (ABA) enhanced ACh- and K-induced tension and simultaneously enhanced their depolarizing actions. Ionophores and ABA appear to enhance calcium influx which may secondarily influence sodium influx. Calcium antagonists have no consistent actions on this muscle, suggesting that calcium channel activity of the radular protractor may be different from that seen in mammalian visceral muscles.