The role of protein kinases in the control of prolonged changes in neuronal excitability.
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Animals↗
Biomedical subjects
L K Kaczmarek
Publications and source records attributed to L K Kaczmarek.
Phorbol esters, protein phosphorylation and the regulation of neuronal ion channels.
Protein kinase C is an enzyme whose activity is modulated by its lipid environment and which is fully activated by diacylglycerol in the presence of phosphatidyl serine and calcium ions. This kinase is highly enriched in the nervous systems of both vertebrates and invertebrates. The activity of protein kinase C can be stimulated in intact cells by certain synthetic diacylglycerols as well as by phorbol esters which substitute for endogenous diacylglycerol. The effects of such activators on the endogenous electrical properties of neurones, as well as on synaptic transmission, have recently been investigated in several vertebrate and invertebrate preparations of neurones. One example is that of the bag cell neurones of Aplysia which, in response to brief stimulation, generate a prolonged discharge during which the height of their action potentials is increased. Exposure of isolated bag cell neurones to activators of protein kinase C results in the enhancement of their action potentials through an increase in the amplitude of their voltage-dependent calcium current. This is caused by the unmasking of a previously inactive species of calcium channel in the plasma membrane.
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Multiple components of delayed potassium current in peptidergic neurons of Aplysia: modulation by an activator of adenylate cyclase.
The neurosecretory bag cell neurons of the mollusk, Aplysia, control egg-laying behavior in the animal. In these cells, elevation of cAMP greatly enhances the height and width of action potentials. A similar enhancement of action potentials is seen during the bag cell afterdischarge, a 30 min period of repetitive activity that may be triggered by peptides from the reproductive tract or by brief extracellular stimulation. The enhancement of action potentials during an afterdischarge is well correlated with the observed elevations of cAMP. In the present study, we have examined the effects of forskolin (an activator of adenylate cyclase) and theophylline (a phosphodiesterase inhibitor) on the delayed outward currents that are likely to control repolarization of the action potential. Isolated bag cell neurons, maintained in primary culture, were studied with a whole-cell patch clamp technique. High intracellular concentrations of EGTA were used to block potassium current activated by calcium entry. Analysis of the remaining voltage-dependent delayed outward current revealed two major components, which could be separated on the basis of their different kinetic properties. Both currents (IK1 and IK2) were carried by potassium. IK1, which did not inactivate during 100 msec depolarizations, was reduced in amplitude by application of forskolin and theophylline. Ik2, a current defined by its faster kinetic properties, partially inactivated during 100 msec depolarizations. This inactivation was markedly speeded by application of forskolin and theophylline. It is suggested that such changes in outward current in response to cAMP could explain the enhancement of spike width seen during an afterdischarge in vivo.
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Inhibitors of calcium-dependent enzymes prevent the onset of afterdischarge in the peptidergic bag cell neurons of Aplysia.
Experiments with isolated bag cell neurons of Aplysia have produced evidence that changes in neuronal excitability may be brought about by the activation of calcium dependent enzymes such as calcium-dependent protein kinases. We have now examined the effects of agents which have been shown to inhibit several calcium-dependent enzymes on the properties of bag cell neurons in situ. In response to brief electrical stimulation the bag cell neurons of Aplysia generate an afterdischarge during which they release neuroactive peptides. We have found that the ability of stimulation to trigger an afterdischarge in the bag cell neurons is inhibited by trifluoperazine (TFP, 50-100 microM), N-(6-aminohexyl)-5-chloro-1-napthalenesulphonamide (W7) (25-50 microM) and calmidazolium (40-100 microM), each of which has previously been shown to inhibit calmodulin-dependent enzymes and the calcium-phospholipid-dependent protein kinase in the bag cell neurons. Further analysis of the effects of TFP showed that this inhibition occurs at concentrations which do not inhibit synaptic transmission or the endogenous bursting of another neurosecretory neuron, R15. Secretion of neuroactive peptides from the bag cell neurons was measured both electrophysiologically and biochemically. No attenuation of secretion could be observed at concentrations of TFP below those which inhibited the initiation of afterdischarge. Our results indicate that these agents inhibit secretion from these neurons primarily by inhibiting the onset of the afterdischarge and are consistent with the hypothesis that a calcium-dependent enzyme plays a role in triggering the stimulus-induced transformation in the electrical properties of these neurons.
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Peptidergic neurons of Aplysia lose their response to cyclic adenosine 3':5'-monophosphate during a prolonged refractory period.
Although the peptidergic bag cell neurons of Aplysia are ordinarily silent, they respond to brief electrical stimulation by producing an afterdischarge of about 30 min duration. This afterdischarge is followed by a refractory period lasting many hours during which electrical stimulation either fails to initiate afterdischarges or produces discharges of much shorter duration. Previous work has demonstrated that cyclic AMP plays a role in the genesis of afterdischarge, both in intact bag cell clusters and in isolated cultured bag cells. We have now examined the hypothesis that in the refractory period either the synthesis of cyclic AMP or the response to cyclic AMP is attenuated. Direct measurements of cyclic AMP showed that cyclic AMP levels in the bag cell neurons are elevated to a similar extent after stimulation in refractory and nonrefractory clusters of neurons. We have found, however, that the response to cyclic AMP is altered during the refractory period. The electrophysiological responses of bag cell neurons were first examined in intact clusters of cells within the abdominal ganglion. Cyclic AMP levels were elevated using the adenylate cyclase activator, forskolin, in the presence of theophylline (FT). The duration of a first bag cell afterdischarge could be greatly increased if FT was added before stimulation. The duration of a stimulated second bag cell afterdischarge could also be significantly increased if FT was added within a brief period following the end of the first afterdischarge. Furthermore, at these times the addition of FT often resulted in the onset of spontaneous afterdischarges.(ABSTRACT TRUNCATED AT 250 WORDS)
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Calcium/phosphatidylserine/diacylglycerol-dependent protein phosphorylation in the Aplysia nervous system.
It has been shown that intracellular injection of protein kinase C (calcium/phosphatidylserine/diacylglycerol-dependent protein kinase), purified from mammalian brain, or application of the tumor-promoting phorbol diester, 12-O-tetradecanoyl-13-phorbol acetate (TPA), leads to an enhancement of calcium currents in the bag cell neurons of Aplysia. We now present evidence of an endogenous enzyme in bag cell neurons which is activated by TPA and which has properties similar to those of mammalian protein kinase C. Calcium/phosphatidylserine/diacylglycerol-dependent protein kinase activity was found in both cytosolic and particulate fractions prepared from isolated clusters of bag cell neurons. This endogenous enzyme phosphorylated an 87,000-dalton protein from bovine brain, which appears to be a specific substrate for protein kinase C, as well as several substrates present in cytosolic fractions prepared from isolated bag cell clusters. Similar results were obtained using preparations made from pooled head ganglia from Aplysia. The pharmacological properties of the calcium/phosphatidylserine/diacylglycerol-dependent protein kinase activity in the Aplysia nervous system were similar to those of protein kinase C from mammalian tissues. Thus, the same group of endogenous substrate proteins were phosphorylated when diacylglycerol was replaced by TPA in cytosolic fractions prepared from isolated bag cell clusters. Non-tumor-promoting phorbols (4-alpha-phorbol, 4-alpha-phorbol-12,13-didecanoate, and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate) did not stimulate protein phosphorylation in these preparations. Phosphorylation by the Aplysia calcium/phosphatidylserine/diacylglycerol-dependent protein kinase was inhibited by polymixin B sulfate, by calmodulin, and by the "calmodulin antagonists" trifluoperazine, calmidazolium and W7.(ABSTRACT TRUNCATED AT 250 WORDS)
Aplysia↗
A voltage-clamp analysis of currents underlying cyclic AMP-induced membrane modulation in isolated peptidergic neurons of Aplysia.
A variety of chemical and electrophysiological evidence indicates that the onset of afterdischarge and the subsequent profound enhancement of spike broadening that occur in the bag cell neurons of Aplysia are related to an increase in adenosine 3',5'-monophosphate-(cAMP) dependent protein phosphorylation. We have now used a two-electrode voltage clamp to study the properties of isolated bag cell neurons in cell culture and their response to 8 benzylthio-cAMP (8BTcAMP) and N6-n-butyl 8BTcAMP. These membrane-permeant and phosphodiesterase-resistant cAMP analogs induce spontaneous discharge and spike broadening in both the intact bag cell cluster and isolated bag cell neurons in cell culture. The dominant inward current in these cultured cells was found to be the calcium current, Ica, which was abolished by Co2+ (20 mM) or Ni2+ (10 mM) and could be observed in Na+-free media. In a minority of cells (2 of 12), in normal ionic media, a transient inward current was observed that was unaffected by Co2+ and Ni2+ and probably represents a sodium current. The three characterized potassium currents, the delayed rectifying current IK, the calcium-dependent current IC, and the early transient current IA, distinguished by their differing pharmacological and voltage-activation properties, were present in all healthy cells. Three effects of the cyclic AMP analogs (0.5 mM) on the electrical properties of these cells were 1) the emergence of a region of negative slope resistance in the steady-state I-V relations, 2) a depression of the net sustained outward currents due to depolarizing commands, and 3) a marked reduction in IA. When outward currents had been largely suppressed using high concentrations of tetraethylammonium (TEA) ions (100-460 mM) no effects of the cyclic AMP analogs could be observed on peak inward currents using NA+ and Ca2+ or Ba2+ as carriers of inward current. At least part of these electrical effects of the cyclic AMP analogs could be accounted for by a depression of a delayed potassium current and the A current.
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Calcium/calmodulin-dependent protein phosphorylation in the nervous system of Aplysia.
An afterdischarge in the bag cell neurons of Aplysia was previously shown to be associated with calcium entry into these cells and with changes in the phosphorylation state of at least two bag cell proteins (BC-I and BC-II). We have now investigated the role of calcium plus calmodulin (Ca/CaM) in the control of phosphorylation of Aplysia nervous system proteins, including those of the bag cell neurons. In cell-free preparations of Aplysia CNS, we demonstrated Ca/CaM-stimulated protein phosphorylation that could be inhibited by the calmodulin-blocking drugs R24571 , trifluoperazine, chlorpromazine, and W7 . A number of substrate proteins for Ca/CaM-dependent protein phosphorylation with Mr values from 17,000 to 310,000 were consistently observed in homogenates of the Aplysia CNS. In the bag cells, we found that a major substrate for Ca/CaM-dependent protein phosphorylation was the bag cell-specific, Mr = 21,000 protein (BC-II). BC-I (Mr = 33,000), on the other hand, appeared not to be a substrate for a Ca/CaM-dependent protein kinase. We found that there are a minimum of two Ca/CaM-dependent protein kinases in the Aplysia nervous system. These enzymes were distinguished on the basis of their subcellular distribution and their ability to phosphorylate distinct sites on synapsin I, an exogenous neuronal protein from vertebrates. Phosphorylation by one of these kinases (calmodulin kinase I) was on a site recovered in an Mr = 10,000 proteolytic fragment of synapsin I, and phosphorylation by the other (calmodulin kinase II) was on a site recovered in an Mr = 30,000 fragment. The predominant enzyme in the Aplysia CNS, as in the mammalian nervous system, was calmodulin kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)
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Calcium entry causes a prolonged refractory period in peptidergic neurons of Aplysia.
A brief train of electrical stimuli to the pleuroabdominal connective of Aplysia produces a cumulative depolarization in the peptidergic bag cell neurons within the abdominal ganglion. This response is followed by an afterdischarge which lasts for about 30 min, and then by a prolonged refractory period lasting for several hours. During the refractory period the cumulative depolarization in response to stimulation is attenuated, and stimulation either fails to initiate afterdischarges or produces discharges of much shorter duration. We have used the cationophore X537A to test the hypothesis that the prolonged refractory period is caused by calcium entry into the bag cell neurons during the afterdischarge. Exposure of intact bag cell clusters to X537A at concentrations from 1 to 10 microM for a period of 20 min in calcium-containing media produced no change in their resting potentials or in their ability to generate action potentials, but induced a state resembling natural refractoriness in response to subsequent stimulation. Both natural refractoriness and that induced by X537A could be overcome by extracellular tetraethylammonium ions (90 mM). Dose response data showed that concentrations of ionophore of 2.5 to 5.0 microM produce an attenuation of afterdischarge that is similar to that following a stimulated afterdischarge. These concentrations of X537A also produced an enhancement of 3H-labeled peptide release from these cells that is comparable to that observed on stimulation of an afterdischarge. Moreover, the time course of recovery from exposure to 5.0 microM X537A parallels that of natural refractoriness, recovery being essentially complete about 20 hr after X537A exposure or stimulation. The ionophore did not affect the mean duration of afterdischarge when applied in calcium-deficient media. The electrical effects of X537A were investigated using isolated bag cell neurons in cell culture. After treatment with the adenylate cyclase activator, forskolin, and theophylline, such isolated cells show many of the electrical changes occurring in intact bag cell clusters at the onset of afterdischarge, including the enhancement of action potentials, as well as increased input resistance and the emergence of oscillations in membrane potential. None of these parameters was significantly affected by concentrations of ionophore that induce refractoriness. In response to repetitive intracellular stimulation, however, some forskolin-treated cells undergo a cumulative depolarization which is similar to that seen at the onset of afterdischarge in intact clusters. This cumulative depolarization was found to be attenuated or abolished by 5.0 microM X537A.(ABSTRACT TRUNCATED AT 400 WORDS)
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An early sodium and a late calcium phase in the afterdischarge of peptide-secreting neurons of Aplysia.
Following brief electrical stimulation of a pleuroabdominal connective nerve, the clusters of peptidergic bag cell neurons of Aplysia generate a long-lasting (or approximately 30 min) synchronous afterdischarge. In a normal seawater medium, the afterdischarge comprises an early phase of rapid firing (2-6 Hz) lasting for less than one minute, followed by a second, prolonged phase of lowering firing rate (less than 0.5 Hz). We have found that the transition from the first, rapid-firing phase to the prolonged second phase of afterdischarge is associated with an increase both in the width and height of intracellularly recorded action potentials. In addition, we find that in the presence of the potassium channel-blocker, TEA, such biphasic afterdischarges may be triggered by depolarizing current that evokes action potentials in a single bag cell neuron in a cluster. Afterdischarges resembling either the first or the second phase of a normal afterdischarge may be produced by using media deficient in calcium or sodium, respectively, in combination with potassium channel-blockers. Brief stimulation in media deficient in calcium give rise to afterdischarges of high firing frequency (2-6 Hz) lasting for less than one minute (mean duration = 41 s) while, in a sodium-free medium, longer afterdischarges (mean duration = 18 min) of lower firing rate (less than 0.3 Hz) were generated. At the end of afterdischarge in a normal seawater medium, containing both sodium and calcium ions, the bag cell neurons become refractory to stimulation. Thus, further stimulation either fails to induce afterdischarge or results in afterdischarges that are attenuated both in frequency of firing and in duration compared with the first afterdischarge. In these experiments we found that the brief afterdischarges, observed in low-calcium media, which resemble the first phase of a normal afterdischarge, did not induce subsequent refractoriness while afterdischarges evoked in the normal calcium, sodium-free medium did result in refractoriness to further afterdischarge. Our data indicate that both phases of a bag cell afterdischarge are due to intrinsic bag cell mechanisms and suggest that the firing pattern in the first phase is largely sodium dependent and that of the second, slow phase is calcium dependent. Moreover, our data indicate that it is the second, calcium-dependent phase that induces the refractoriness that follows bag cell afterdischarge.
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Protein phosphorylation during afterdischarge in peptidergic neurons of Aplysia.
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Aplysia↗
Intracellular modulation of membrane channels by cyclic AMP-mediated protein phosphorylation in peptidergic neurons of Aplysia.
The peptidergic bag cell neurons of the opisthobranch mollusc Aplysia control egg laying and its correlated behavior by release of the neuroactive peptide, egg-laying hormone, during the extended electrical discharge termed afterdischarge. This paper examines the evidence for the involvement of cyclic AMP (cAMP) and protein phosphorylation in the mediation of this electrical afterdischarge. It is concluded that an important component in the mechanism of afterdischarge is the suppression of a potassium channel, mediated by cAMP-dependent protein kinase-induced protein phosphorylation. The exact identity of the potassium channel remains to be worked out.
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Serotonergic inhibition of afterdischarge in peptidergic bag cells.
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The expression of long lasting afterdischarge by isolated Aplysia bag cell neurons.
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Microinjection of catalytic subunit of cyclic AMP-dependent protein kinase enhances calcium action potentials of bag cell neurons in cell culture.
We have found that the calcium action potentials of bag cell neurons from the abdominal ganglion of Aplysia may be enhanced by intracellular microinjection of the catalytic subunit of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). The catalytic subunit was purified from bovine heart and shown to be effective in stimulating the phosphorylation of bag cell proteins in homogenates at concentrations of 10-50 nM. Intracellular injection into isolated bag cell neurons maintained in primary culture was through pressure applied to microelectrodes filled at the tip with catalytic subunit (5-22 muM). In 11 of 16 injected cells, both the slope of the rising phase and the height of the action potentials evoked by a constant depolarizing current were markedly enhanced relative to the pre-injection control (mean increases, 73% and 35%, respectively). This effect could occur with no change in resting potential or in the latency of the action potential from the onset of the depolarizing pulse. The effect was observed with enzyme dissolved in three different salt solutions (Na phosphate, K phosphate, or KCl). In two experiments, tetrodotoxin (50 muM) added to the extracellular medium had no effect on the enhanced action potentials. Subsequent addition of the calcium antagonist Co(2+), however, diminished or abolished the spikes. In more than half of the experiments, the injection of catalytic subunit was accompanied by an increase in the input resistance of the cells as measured by applying small hyperpolarizing current pulses. In three experiments, subthreshold oscillations in membrane potential resulted from the injections. Control injections (24 cells), carried out either with carrier medium alone or with heat-inactivated enzyme preparations, did not produce spike enhancement, increased input resistance, or oscillations. Our data suggest that the stimulation of intracellular protein phosphorylation by the catalytic subunit of cyclic AMP-dependent protein kinase enhances the excitability of bag cell neurons by modifying calcium and potassium channels or currents.
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Purification and primary structure of two neuroactive peptides that cause bag cell afterdischarge and egg-laying in Aplysia.
Two neuroactive peptides, A and B, have been isolated from the atrial gland in the reproductive tract of Aplysia. Each of the two peptides is able to induce egg-laying behavior in recipient animals. In vitro recordings from the abdominal ganglion show that both peptides also trigger longlasting discharges in the bag cell neurons at concentrations around 0.1 muM. The peptides were purified by a combination of ammonium sulfate precipitation, agarose gel filtration, and cation exchange chromatography. Each peptide has 34 amino acid residues. Microsequencing together with carboxypeptidase Y degradation and analysis of tryptic peptides revealed the following sequence for peptide A: H-Ala-Val-Lys-Leu-Ser-Ser-Asp-Gly-Asn-Tyr-Pro-Phe-Asp-Leu-Ser-Lys-Glu-Asp-Gly -Ala-Gln-Pro-Tyr-Phe-Met-Thr-Pro-Arg-Leu-Arg-Phe-Tyr-Pro-Ile. Peptide B differs from A in only four positions. The first nine residues of B are: Ala-Val-Lys-Ser-Ser-Ser-Tyr-Glu-Lys-, whereas residues 10-34 of B are identical to those of A. The calculated M(r) of A is 3924 and that of B is 4032. The pI of peptide A as determined by isoelectric focusing in polyacrylamide gels is 7.9-8.1 and that of peptide B is 9.0-9.2. It is estimated that each atrial gland contains at least 150 mug of peptide A and 50 mug of B. Neither peptide resembles the egg-laying hormone isolated from bag cell neurons. It is postulated that the atrial gland peptides are released during copulation, and then by interacting with neuronal receptors in the head ganglia and pleuroabdominal connectives they cause the bag cells to afterdischarge, thereby releasing egg-laying hormone.
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Peptides controlling behavior in Aplysia.
Figure 11 summarizes our present understanding of the relationships between the bag cells, the atrial gland, their respective peptides, the central nervous system, and reproductive behavior. There are some interesting aspects of the overall organization of the system. The three hormonal peptides (ELH and the two atrial gland peptides) have specific actions on the central nervous system not unlike what we are currently learning from mammalian systems (e.g., LHRH and TRH). ELH, in addition, has several specific peripheral targets, the details of which remain to be worked out. The fact that ELH and other hormones have multiple targets within the central nervous system as well as nonnervous peripheral targets raises the question of whether one or more different receptors exist for single hormone. We suggest that peptides larger than perhaps five residues may carry several "messages" or receptor binding sites encoded within the one molecule. Large peptides such as ELH could obviously have separate domains of charge distribution within the molecule, and these would have the advantage, over the classical small molecule transmitters, of activating a variety of very different targets. The atrial gland is a peripheral source of peptides with potent nervous system actions; this is reminiscent of peptides in mammals, e.g., substance P, gastrin, and somatostatin, all of which were initially isolated from the gut and which are now being found in and also have actions on the central nervous system. Such resemblances in the principles of organization between mammals and molluscs are constant reminders that neuropeptidergic systems are old tricks in the evolutionary bag and that what we learn from molluscs and other invertebrates about mechanisms and organization will likely apply to mammals.
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The morphology and coupling of Aplysia bag cells within the abdominal ganglion and in cell culture.
The bag cells in the abdominal ganglion of Aplysia californica control egg-laying behavior by releasing a polypeptide (ELH) during an afterdischarge of synchronous action potentials. We have used intracellular injection of Lucifer Yellow to study the morphology and interconnections of the bag cells. These neurosecretory cells are typically multipolar and their processes extend in all directions out from the bag cell clusters into the surrounding connective tissue, where they branch in a complex manner. In some of the dye injection experiments, dye transfer from the injected cell to neighboring cells was observed. Freeze fracture of the bag cell clusters and their surrounding connective tissue revealed numerous gap junctions on bag cell processes within the clusters as well as on more distal processes. We have also examined the morphology and coupling between bag cells in primary culture. As in the intact ganglion, bag cells in culture were found to be multipolar. All pairs of bag cells whose somata or processes had formed contacts in culture were electrically coupled. The strongest coupling was observed between pairs of cells whose somata appeared closely apposed. In these cases transfer of Lucifer Yellow between cells could also be observed. It is therefore likely that the synchrony of bag cell action potentials during a bag cell afterdischarge is a result of coupling between individual cells in the bag cell cluster.
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