Inhibition of peptide release from invertebrate neurons by the protein kinase inhibitor H-7

Brain Research, 581 (1992) 315-318 © 1992 Elsevier Science Publishers B.V. All fights reserved. 0006-8993/92/$05.00

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Inhibition of peptide release from invertebrate neurons by the protein kinase inhibitor H-7 Karen J. Loechner, Joanne Mattessich-Arrandale, Edward M. Azhderian and Leonard K. Kaczmarek Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510 (USA)

(Accepted 11 February 1992) Key words: Protein kinase C; Egg-laying hormone; Aplysia; Bag cell neuron; Calcium channel

The protein kinase inhibitor H-7 has been shown to prevent the potentiation of action potentials that normally accompanies an afterdischarge in the bag cell neurons of Aplysia. We have now shown that H-7 attenuates the release of ELH from these neurons during an afterdischarge without influencing the firing frequency or length of the afterdischarge. The timing and frequency of egg-laying behaviors in Aplysia is controlled by the bag cell neurons, located in the abdominal ganglion of this animal. Because these neurons undergo long lasting changes in their electrical properties, they have been used as a model system for the investigation of mechanisms that regulate ion channels and the relationship between electrical and secretory properties of neurons. Upon brief electrical or hormonal stimulation, these neurons depolarize and fire repetitively for a period of approximately 20-30 min 14' 21. During this afterdischarge, several neuroactive peptides are released from varicosities along the axons of bag cell neurons into the surrounding vascularized connective tissue sheath 5. These peptides include egg-laying hormone (ELH) 1'2'24'27, a 36-amino acid peptide that, when injected in vivo, can induce the fixed action pattern of egg-laying 1'4'26. Within minutes after the onset of an afterdischarge, there is an increase in inositol phosphate turnover 11 and the action potentials of bag cell neurons increase in height and width 18. Similar enhancement of action potentials can be observed in isolated bag cell neurons following treatment with activators of protein kinase C such as phorbol esters, diacylglycerol, or direct microinjection of protein kinase C into the cells 8'28. The mechanism underlying the potentiation appears to involve an increase in the amplitude of voltage-dependent calcium current caused by the recruitment of a new species of calcium channel that is not detected in the plasma membrane prior to activation of protein kinase C 28. Previous work has shown that phorbol ester-induced

changes in voltage-dependent calcium current are prevented by pre-treatment of bag cell neurons with the protein kinase inhibitor H-7 (1-(5-isoquinolinesulfonyl)2-methyl piperazine) 7. Moreover, the enhancement of action potentials that follows electrical stimulation of an afterdischarge is also attenuated by H-77. Although pharmacological inhibitors of protein kinases such as H-7 are often highly selective for a specific pathway l°'tS't9, Conn et al. 6 have shown that H-7 is relatively selective for protein kinase C over the cyclic AMP-dependent protein kinase in the bag cell neurons. For example, H-7 does not inhibit the effects of cyclic AMP analogs or forskolin, an activator of adenylate cyclase, on 32p-incorporation into phosphoproteins, movement of secretory granules, or modulation of voltage-dependent delayed potassium currents in these neurons. Given the relative selectivity of H-7 for protein kinase C over protein kinase A in the bag cell neurons and its effects on the potentiation of action potentials, we have now tested the effects of this inhibitor on the release of egg-laying hormone from these neurons. Electrophysiology and peptide collection were carried out as described by Loechner et al. 22. Briefly, adult animals (300-600 g) were anesthetized by injection of isoosmotic MgC12 equivalent to half of body weight prior to dissection in order to prevent premature afterdischarges. Abdominal ganglia, including bag cell clusters, pleurovisceral connective nerves, and artery to the abdominal ganglia were removed and placed in a 1-ml dish containing equal volumes of isoosmotic MgC12 and artificial sea water (ASW) (in mM: NaCI 460, KCI 10.4,

Correspondence: L.K. Kaczmarek, Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.

316 A

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1,5

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u ASW t, H-7

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uo 0.5

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ELH *

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Time (minutes)

410

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TIME (min) Fig. 1. A: extracellular recording of a control (ASW) afterdischarge in bag cell neurons following electrical stimulation (S). Only the first 10 min of the afterdischarge are shown. B: extracellular recording of the onset of an afterdischarge from a ganglion pretreated with H-7 (75 #M). The fast phase of firing in this experiment occurred twice during the first few minutes of the afterdischarge. Such a pattern can be seen in both ASW- and H-7treated ganglia and represents the slight asynchrony of firing between the two electronically coupled bag cell clusters 3. C: HPLC chromatogram illustrating the separation of ELH and non-ELH peaks.

H E P E S 15, CaCI2 11, and MgCI2 55; p H = 7.8). The artery to the abdominal ganglion was cannulated as described by Mayeri et al. 23. Following cannulation, the m e d i u m surrounding the ganglion was replaced with ASW. Afterdischarges were stimulated with a suction electrode placed over the proximal pleurovisceral connective nerve (8-20 V, 6 Hz, 2.5 ms pulse width, 5 s duration). Bag cell electrical activity was recorded with a suction electrode placed over the ipsilateral bag cell cluster. The artery was perfused with

5 ....1

w ~v 4

lib

Fig. 3. The mean release of ELH during each 5 min period of electrical activity is shown for both ASW-, ([~) and H-7- (A) treated ganglia. The numbers next to the symbols represent the number of samples used at each time point. The change in these numbers over time reflects the distribution of afterdischarge durations.

either A S W (control) or the protein kinase inhibitor H-7, at 9/A/min beginning at least 30 min prior to stimulation and continuing throughout t h e afterdischarge. The m e d i u m surrounding the ganglion paralleled that in the cannula. H-7 was obtained from Seigagaku A m e r i c a (FL) and was dissolved in distilled water to m a k e a stock solution of 10 m M H-7 and then diluted in A S W to a final concentration of 50-75 # M prior to experiments. Following stimulation of the afterdischarge, the medium surrounding the ganglion was completely exchanged at 5 min intervals during the course of the afterdischarge. All samples were stored at -20°C until analyzed using high-pressure liquid c h r o m a t o g r a p h y (HPLC). The integrity of the cannulation and arterial perfusion was ascertained at the conclusion of each exp e r i m e n t by injection of the dye, Tryphan blue. Peptides in the superfusing m e d i u m were separated by H P L C using a Vydac reverse phase C18 column (5/~M, 300 •, 2.5 × 15 cm) and a segmental linear gradient of 0.05% trifiuoroacetic acid in acetonitrile (5-39% acetonitrile in 39 min). Post-column derivitization with o-phthalicdicarboxaldehyde 25 was then used and the peptides detected with a Hitachi F-1600 s p e c t r o p h o t o m e t e r . E L H was identified by coelution with a synthetic peptide standard (Peninsula Labs, Inc., Belmont, C A ) and by amino

c'-

~~

2 TABLE I 1

0

Effect of H-7 on the afterdischarge

ASW (11)

H-7

Group

Mean duration of afterdischarge (rain)

Mean frequency (spikes~rain)

ASW (n = 11) H-7 (n = 8)

25.00 + 2.27 19.62 + 1.79

27.19 _+ 1.99 22.98 +_ 1.61

(8)

*p < 0.005 Fig. 2. Histogram demonstrating the inhibition of ELH release during afterdischarges in control (ASW)- and H-7-treated ganglia.

317 acid analysis 22. Fig. 1C shows a representative chromatogram showing the separation of E L H and non-ELH peaks. Levels of E L H were quantified by measuring peak amplitudes and comparing these to the amplitude of the peak for the synthetic standard. Fig. 1 shows extracellular recordings of electrically stimulated afterdischarges in clusters of bag cell neurons in control medium (A) or medium containing H-7 (75 /~M) (B). Both afterdischarges exhibit an initial fast phase, during which time the neurons fire at approximately 4-6 Hz, followed by a slower phase during which the average firing frequency is approximately 0.5 I/z, as has been described by Kaczmarek et al. TM. Fig. 2 shows the inhibition of E L H release during an aflerdischarge in the presence of H-7. The mean total release of E L H in ganglia treated with H-7 (50-75/~M) was 0.93 + 0.40 btg (n = 8) as compared to 4.18 + 0.82/~g in ASW (n = 11). Student's t-test showed that the effect of H-7 was significant at P < 0.005. We also examined in more detail the profile of release of E L H over the course of the afterdischarge. Fig. 3 shows the mean amount of ELH released during each 5 min interval of electrical activity from afterdischarges of varying durations. As can be seen, the amount of E L H released in ganglia perfused with ASW was greater than that released in H-7-treated ganglia at each interval during the afterdischarge. The difference in release was significant at the 10, 15, and 20 min collection times during the afterdischarge (P < 0.05). To determine whether H-7 could be attenuating the secretion of E L H by inhibiting either the rate of firing during afterdischarges or the duration of the afterdischarges, we measured these parameters in the ganglia in which release had been quantified. Neither of these parameters were significantly altered by H-7 treatment (see Table I). This is in agreement with previous work on the electrophysiological effects of H-7 on bag cell neurons 7. Moreover, no consistent variations in firing patterns (e.g.

intensity or timing of bursts) that might affect release 9 were seen after treatment with H-7 (see Fig. 1). Our results demonstrate that H-7, a relatively selective inhibitor of protein kinase C over protein kinase A in bag cell neurons, can attenuate release of ELH during an afterdischarge. Although we cannot exclude the possibility that H-7 inhibits kinases or enzymes other than protein kinase C in the bag cell neurons, the results are consistent with a role for protein kinase C in the modulation of peptide release. It is likely that H-7 attenuates secretion by inhibiting the potentiation of calcium action potentials that normally occurs during the afterdischarge 7. This would be expected to diminish calcium influx and thereby attenuate calcium-dependent exocytosis. For example, Fossier et al. 12 found that H-7 blocked the protein kinase C-mediated potentiation of evoked quantal release of acetylcholine by FLRFamide in Aplysia buccal ganglia. The authors proposed that H-7 inhibited the facilitation of calcium current that normally occurred in response to FLRFamide. Alternatively, H-7 may also inhibit the action of enzymes such as protein kinase C on the secretory apparatus itself, directly diminishing the rate of exocytosis of ELH-containing granules. Protein kinase C has been implicated in the regulation of exocytosis in a variety of systems, including the release of catecholamine from adrenal chromaffin cells ~7' 20,29 and from hippocampus 16 as well as potassiumevoked release of acetylcholine from Torpedo electric organ synaptosomes ~3. Furthermore, the potentiation of acetylcholine release by activators of protein kinase C was found to be blocked by H-7 t3. In Aplysia, regulation of the secretion of ELH and other bag cell peptides by second messenger pathways in vivo may play a role in determining whether or not egg-laying behavior will be triggered by stimulation of afferent inputs to the bag cell neurons.

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This work was supported by NIH Grant NS18492 to L.K.K.

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