SUMMARY1. Current-and voltage-clamp ,methods were used to evaluate the intracellular and ionic mechanisms involved in dopamine-induced slow depolarizations recorded from neurones of the LB cluster in the abdominal ganglion of Aplysia kurodai.2. In voltage-clamped cells, dopamine induced a slow inward current that, over the range studied (-40 to -110 mV), decreased in amplitude with hyperpolarization of the cell, but failed to invert when the cell was hyperpolarized beyond the reversal potential for K+, EK.3. Bathing the ganglion in 3-isobutyl-1-methylxanthine (IBMX) caused a significant increase in the dopamine response.4. Most of the responses to dopamine were markedly augmented in Ca2+-free media, but were depressed in Na+-free media.5. An intracellular injection of cyclic adenosine 3',5'-monophosphate (cyclic AMP) into the same cell type produced an inward current which, like the response to dopamine, diminished in amplitude with hyperpolarization of the cell.6. Like the dopamine response, the cyclic AMP response increased in the presence of IBMX, was enhanced in Ca2+-free media, was depressed in Na+-free media, and was unaffected by changes in external potassium.7. In a few cells, although the cyclic AMP-induced responses disappeared in Na+-free media, the dopamine-induced slow inward current responses did not. However, these Na+-free resistant responses disappeared completely in Na+-and Ca2+-free media.8. It was concluded that most of the dopamine-induced inward current responses were produced by an increase in permeability, mainly to Na+, triggered by a receptor-controlled increase in intracellular cyclic AMP.

The follicular cells of Xenopus laevis are known to have various types of receptors such as ␤-adrenergic-[1], muscarinic [2,3], , , adenosine- [8][9][10], and purinergic receptors [3,[11][12][13] in their cytoplasmic membranes. Among the agonists (transmitters or hormones) for these receptors, noradrenaline and ATP are released from sympathetic nerve endings [14], and acetylcholine is released from parasympathetic nerve endings. FSH and LH are released from the hypophysis and delivered via circulating blood. The blood is also known to contain several M of adenosine and ATP [15]. When these agonists stimulate their receptors, they trigger the activation of various intracellular enzyme systems, which subsequently synthesize intracellular second messengers such as cyclic adenosine 3Ј,5Ј-monophosphate (cAMP), diacylglycerol (DAG), and inositol 1,4,5-triphosphate (IP 3 ). These second messengers initiate or modulate various kinds of intracellular signaling of the cells, which are considered necessary for oocyte maturation.Xenopus follicles consist of a single large oocyte surrounded by a monolayer of small follicle cells attached to the oocyte by gap junctions. The gap junctions have connexin channels, which can permeate various molecules and ions smaller than a molecular weight of 1,000, including second messengers such as cAMP, IP 3 , and Ca

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Application of either acetylcholine (ACh), dopamine (DA), histamine (HA), or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces a K+-current response in the identified neurons of Aplysia under voltage clamp. This type of response is mediated by a pertussis toxin (PTX)-sensitive G-protein, Gi or Go. Extracellular application of 60 microM phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), to these cells markedly depressed all the K+-current responses to ACh, DA, HA, and FMRFamide. The depressing effect of PDBu lasted for at least 60 min despite continuous washing with the normal perfusing medium. Application of PKC inhibitors such as 100 microM H-7 or 10 microM staurosporine and PKCI(19-31) prior to the application of PDBu significantly decreased the depressing effects of PDBu. In contrast, an intracellular injection of okadaic acid (OA), an inhibitor of protein phosphatase 1 and 2A, significantly augmented the blocking effect of PDBu. Intracellular injection of the PKC catalytic subunit induced a similar depressing effect as observed with PDBu. The dose-response curves obtained with different transmitters all shifted downward after the activation of PKC, but the ED50 of each transmitter remained unchanged. Furthermore, the K+-current responses induced by the intracellular application of GTPgammaS were not depressed at all, even after the receptor-induced K+-current responses of the same cell were markedly depressed. These results strongly suggest that PKC phosphorylated a certain coupling site between the receptor and G-protein, and impaired the signal transduction necessary for triggering the K+-channel opening.

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