Inhibitors of protein kinase C prevent enhancement of calcium current and action potentials in peptidergic neurons of Aplysia

Following brief stimulation of an afferent pathway, the bag cell neurons of Aplysia undergo a dramatic change in excitability, resulting in a prolonged discharge of spontaneous action potentials. During the discharge, the action potentials of the bag cell neurons become enhanced in height and width. The afterdischarge triggers release of neuroactive peptides that initiate egg-laying behavior in this animal. Evidence suggests that changes in excitability of the bag cell neurons may be mediated by activation of protein kinase C (PKC) and cAMP-dependent protein kinase (cAMP-PK). PKC activators, such as the phorbol ester TPA (12-O-tetradecanoyl-13-phorbol acetate), enhance the amplitude of action potentials in isolated bag cell neurons in cell culture. These agents act by unmasking a previously covert species of voltage-dependent calcium channel resulting in an increase in calcium current. In the accompanying paper (Conn et al., 1989), we showed that H-7, a protein kinase inhibitor, inhibits the effect of TPA, and is a selective inhibitor of PKC relative to cAMP-PK in these cells. We now report that another PKC inhibitor, sphinganine, also inhibits the effect of TPA on action potential height and calcium current in cultured bag cell neurons, and that N-acetylsphinganine, an inactive sphinganine analog, fails to inhibit the effects of PKC activators. Although both H-7 and sphinganine prevent the effects of TPA when added prior to TPA addition, neither compound reverses the effects of TPA when added after the action potentials have already become enhanced by TPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phorbol esters: voltage-dependent effects on calcium-dependent action potentials of mouse central and peripheral neurons in cell culture

The beta-phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu), which activate protein kinase C, were applied to mouse dorsal root ganglion (DRG) and cerebral hemisphere neurons grown in primary dissociated cell culture. Phorbol esters did not modify the membrane potential or input resistance of either type of neuron. To assess the effects of beta-phorbol esters on voltage-dependent conductances, the effects of PDBu and TPA on action potentials evoked from these neurons were determined. The neurons were bathed in a solution containing 5 mM tetraethylammonium and action potentials that contained sodium and calcium components were evoked. When applied at resting membrane potential and at more negative potentials, PDBu and TPA reversibly increased action potential duration. The alpha-phorbol ester 4-alpha-phorbol, which does not activate protein kinase C, did not modify action potential duration. The effects of the beta-phorbol esters, however, were voltage-dependent. When the neurons were depolarized to membrane potentials less negative than -50 mV, PDBu and TPA reduced action potential duration. The effects of both PDBu (10 nM-1 microM) and TPA (100 pM-100 nM) on action potential duration were dose-dependent. The prolongation of action potentials produced at large negative potentials may be due to a reduction in voltage-and/or calcium-dependent potassium conductance, since the prolongation was associated with a reduction in the potassium-dependent afterhyperpolarization; following membrane depolarization in control solution, action potential duration was increased for several minutes, while the afterhyperpolarization was reduced and, following this prolongation, phorbol esters no longer prolonged the action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Second messengers involved in the mechanism of action of bradykinin in sensory neurons in culture

Application of bradykinin to neonatal rat dorsal root ganglion neurons caused a depolarization associated with an inward current and an increase in membrane conductance that was probably due to the opening of sodium channels. No hyperpolarization or outward current was detected. In addition, bradykinin increased the rate of 45Ca uptake into the neurons by a mechanism that was blocked by the dihydropyridine calcium channel antagonist nifedipine. Direct activation of protein kinase C (PKC) with phorbol esters mimicked the ability of bradykinin to depolarize the neurons and to increase the rate of 45Ca uptake. Down-regulation of PKC by prolonged treatment with phorbol esters and treatment of the cells with staurosporine, which inhibits PKC, blocked both bradykinin- and phorbol ester-induced 45Ca influx, and substantially reduced the proportion of cells that gave electrophysiological responses to either agent. Bradykinin also activated polyphosphoinositidase C in the dorsal root ganglion neurons, elevating levels of inositol(1,4,5)-trisphosphate and 1,2-diacylglycerol, an endogenous activator of PKC. It is suggested, therefore, that PKC may mediate some of the effects of bradykinin in sensory neurons.     

Alpha bag cell peptide directly modulates the excitability of the neurons that release it

Brief electrical or hormonal stimulation of the bag cell neurons of Aplysia triggers a long-lasting discharge during which alpha bag cell peptide (alpha-BCP) and other neuropeptides are released from the cells. We have carried out experiments, using both intact abdominal ganglia and isolated neurons, demonstrating that alpha-BCP acts directly on the bag cell neurons to influence cAMP levels and voltage-dependent potassium currents. Exposure of the bag cell neurons within intact ganglia to alpha-BCP, at concentrations greater than 1 nM, inhibited an ongoing discharge. alpha-BCP also significantly reduced both basal and forskolin-stimulated levels of cAMP in bag cell clusters. The inhibition of the discharge by alpha-BCP could be prevented and reversed by pharmacological elevation of intracellular cAMP levels. Immunohistochemical staining of neurons maintained in cell culture showed that all isolated bag cell neurons exhibit immunoreactivity with antisera against alpha-BCP. Application of the adenylate cyclase activator forskolin to such isolated cells, in the presence of a phosphodiesterase inhibitor, attenuates the amplitude of the delayed voltage-dependent outward currents measured in voltage-clamp experiments. Pretreatment of the cells with alpha-BCP significantly reduced the ability of forskolin to attenuate these currents, demonstrating that alpha-BCP acts directly at autoreceptors on bag cell neurons. Experiments with the isolated cells showed that a second autoreceptor-mediated effect of alpha-BCP was the enhancement of an inwardly rectifying potassium current that was activated at potentials more negative than -40 mV. The reversal potential and conductance of the current induced by alpha-BCP were dependent on the external K+ concentration. This response to alpha-BCP could be blocked by rubidium, cesium, and barium ions. Our data demonstrate that alpha-BCP can exert inhibitory biochemical and electrophysiological actions on the bag cell neurons that release it and suggest that autoreceptors for alpha-BCP play an important role in the termination of a discharge in the bag cell neurons.     

Inhibition of protein kinase C activity promotes the neurotrophic action of epidermal and basic fibroblast growth factors

In primary neuronal cultures the activation of protein kinase C (PKC) by tumor-promoting phorbol esters blocked growth factor-induced neuronal survival and neurite extension. Depletion of PKC markedly facilitated both epidermal growth factor and basic fibroblast growth factor-inducible neurite extension. Inhibition of PKC by H-7 also stimulated neurite extension. These results suggest that down-regulation of PKC in neurons may be required for trophic factor action.     

Protein kinase C activators block specific calcium and potassium current components in isolated hippocampal neurons

Whole-cell voltage-clamp techniques were used to study the effects of the protein kinase C (PKC) activators phorbol esters and OAG on Ca and K currents in differentiated neurons acutely dissociated from adult hippocampus and in tissue-cultured neurons from fetal hippocampus. PKC activators had selective depressant effects on K currents, with persistent currents (IK and IK-Ca) being reduced and transient current (IA) being unaffected. In both cell types we recorded both high-voltage-activated, noninactivating (L-type) and high-voltage-activated, rapidly inactivating (N-type) Ca current. A low-voltage-activated, rapidly inactivating (T-type) Ca current was also recorded in tissue-cultured neurons but not in acutely dissociated neurons. PKC activators markedly reduced N-type current with less effect on L-type and no effect on T-type Ca current. Effects of PKC activators could be reversed with washing or with application of PKC inhibitors H-7 or polymyxin-B, an effect that could not be attributed to inhibition of cAMP-dependent protein kinase. The Ca/calmodulin inhibitor calmidazolium was ineffective in reversing the actions of PKC activators. Using whole-cell voltage-clamp techniques, we have demonstrated that hippocampal neurons possess 3 distinguishable components of calcium current. Distinct K currents were also observed. Our data strongly support the hypothesis that both Ca and K currents are selectively regulated by PKC and that these effects occur directly on the postsynaptic neuron.     

Long-term depression of a sympathetic ganglionic response to opioids by prolonged synaptic activity and by phorbol esters

We studied the effect of the adenylate cyclase activator forskolin, of protein kinase C-activating phorbol esters and of prolonged preganglionic input activation on the inhibitory response of the perfused superior cervical ganglion of the cat to exogenous met-enkephalin (Met-ENK). Met-ENK inhibited, in a concentration-dependent manner, the postganglionic compound action potential evoked by cervical sympathetic trunk stimulation. The inhibition was reversible, was blocked by naloxone as well as by pertussis toxin and showed no homologous desensitization in the concentration range 0.01–10 μM. Pretreatment of the ganglion with 4β-phorbol 12,13-dibutyrate or 4β-phorbol 12,13-diacetate depressed the Met-ENK response for several hours, while pretreatment with forskolin had no effect. This action of phorbol esters was prevented by the protein kinase inhibitor H-7 but not by the calmodulin antagonist W-7 or the protein kinase A inhibitor HA 1004 and was calcium-dependent. Recovery of the response from the depression produced by phorbol esters was not affected by a protein synthesis inhibitor. A 40 Hz 20 min stimulus train to the cervical sympathetic trunk mimicked the effect of phorbol esters, depressing for several hours the inhibition produced by Met-ENK. Stimulus trains of duration shorter than 5 min or frequency lower than 5 Hz were ineffective. This effect of prolonged preganglionic stimulation occurred even when the stimulus train was delivered during complete block of nicotinic and muscarinic ganglionic transmission but was lost when the stimulus train was delivered during perfusion with calcium-free Krebs. The protein kinase inhibitor H-7 prevented the depression of the Met-ENK response by the train, while W-7 and HA 1004 had no effect. These findings suggest that, in the superior cervical ganglion of the cat, a kinase, activated by phorbol esters and inhibited by H-7, exerts a long-term control of the ganglion cell responsiveness to opiate receptor activation. A similar mechanism can be synaptically activated by a non-cholinergic transmitter, released by the preganglionic axons during prolonged, high frequency, activity.     

Phorbol esters mimic some cholinergic actions in hippocampal pyramidal neurons

Muscarinic receptor stimulation in the hippocampus has been associated with inositol phospholipid breakdown. In other systems this leads to the formation of inositol trisphosphate and diacylglycerol, which promotes the activation of protein kinase C. Phorbol esters, which directly activate protein kinase C, exhibit high and specific binding in the hippocampus. This, along with the advantages of the hippocampal slice preparation, including direct pharmacological access to a cell population (CA1 pyramidal cells) having clearly defined muscarinic responses, makes this an ideal preparation to examine whether protein kinase C serves as the intracellular signal for muscarinic receptor occupation. Like muscarinic agonists, phorbol esters abolish the slow calcium-activated potassium afterhyperpolarizing potential (AHP) and its underlying current without reducing calcium action potentials. Those phorbol analogs that do not activate kinase C have no effect, suggesting that activation of this enzyme is required to reduce the AHP. The accommodation of spike discharge normally seen during a long depolarizing stimulus is also markedly reduced by phorbol esters as well as by muscarinic receptor activation. However, unlike muscarinic agonists, phorbol esters have no effect on the muscarine-sensitive, voltage-dependent, potassium current termed IM, nor do they consistently cause an increase in input resistance. Moreover, unlike ACh, they do not appear to have a presynaptic inhibitory action on the fast EPSP elicited by orthodromic stimulation. The slow cholinergic EPSP was blocked by phorbol esters, but this could be accounted for by a postsynaptic action. Thus, if inositol phospholipid turnover is involved in mediating muscarinic responses in the hippocampus, the activation of protein kinase C can account for only part of the electrophysiological response.     

Down regulation of protein kinase C in neuronal cells: effects on neurotransmitter release

We investigated the effects of phorbol esters on protein kinase C (PKC) activity and on neurotransmitter release from cultured neuronal cells. Both differentiated and undifferentiated PC12 pheochromocytoma cells contained high levels of protein PKC. Under normal conditions all the enzyme activity was found in the cytoplasm. Addition of the phorbol esters phorbol 12-myristate-13-acetate (TPA) or phorbol 12,13-dibutyrate (PDBu) caused a rapid translocation of PKC from the cytoplasm to the particulate fraction. Continued culture of cells with these phorbol esters resulted in the decline of total PKC activity. After 10-20 hr of culture, both membrane and cytoplasmic PKC activity had declined to background levels. cAMP-dependent and Ca2+/calmodulin-dependent protein kinase activities were only slightly affected by chronic phorbol ester treatment. Addition of active phorbol esters to PC12 cells produced an enhancement of the depolarization-induced release of 3H-norepinephrine. Following chronic phorbol ester treatment, the ability of these substances to enhance evoked catecholamine release was lost. Furthermore, depolarizing stimuli released considerably less 3H-norepinephrine than in control untreated cells. Phorbol esters also enhanced depolarization-induced 3H-norepinephrine release from primary cultures of rat sympathetic neurons. Chronic treatment of these neurons with phorbol esters also resulted in the loss of their ability to enhance transmitter release and in a large reduction in the extent of depolarization-evoked transmitter release. Chronic phorbol ester treatment also resulted in the disappearance of PKC from sympathetic neurons, but had little effect on cAMP-dependent or Ca2+/calmodulin-dependent kinase activities. These results demonstrate that PKC-deficient neurons can be prepared. The data also demonstrate that depolarization-induced neurotransmitter release is mediated by both protein kinase C-dependent and independent pathways.     

Protein kinase C mediates potentiation of synaptic transmission by phorbol ester at parallel fibers in the dorsal cochlear nucleus

Many cells in the outer two layers of the dorsal cochlear nucleus (DCN) express high levels of the phospholipid-activated, calcium dependent kinase, protein kinase C (PKC), an enzyme that can phosphorylate numerous proteins involved in neurotransmission and postsynaptic signaling. We investigated the effects of stimulating PKC with phorbol esters (phorbol 12-13 diacetate; PDAc) on parallel fiber synaptic transmission in brain slices of the guinea pig DCN. Phorbol esters increased the amplitude of the postsynaptic components of the field potential, including the excitatory post-synaptic field potential (fEPSP) and the population spike following electric stimulation of parallel fibers. Phorbol esters simultaneously decreased paired-pulse facilitation, suggesting that transmitter release mechanisms were affected. Potentiation of synaptic transmission and diminished paired-pulse potentiation were also observed in intracellular recordings of DCN neurons. The effects of phorbol esters were antagonized by the specific PKC blockers bisindolylmaleimide and calphostin C. Although modulation of the synaptic potentials appears to be mediated by presynaptic PKC, the differential effects of PDAc on the fEPSP and the population spike also suggest the involvement of postsynaptic PKC and postsynaptic targets. These experiments demonstrate that protein kinase C is capable of profoundly modulating synaptic transmission at parallel fiber synapses in the DCN.     

Investigation of PKC isoform-specific translocation and targeting of the current of the late afterhyperpolarizing potential of myenteric AH neurons

AH neurons in the enteric nervous system play an essential role in initiating intestinal reflexes and factors that control AH neuron excitability therefore influence the state of the digestive system. Prominent afterhyperpolarizations that follow action potentials in these neurons strongly affect their excitability. In the present work, we have investigated the regulation of the afterhyperpolarizing current (I(AHP)) by protein kinase C (PKC). Electrophysiological responses and protein translocation were investigated in AH neurons of freshly dissected preparations of myenteric ganglia from the guinea-pig ileum. The activator of conventional and novel PKCs, phorbol dibutyrate, but not the activator of novel PKCs, ingenol, blocked the I(AHP). Phorbol dibutyrate had no effect on the hyperpolarization-activated current (I(h)) or on the A current (I(A)). Stimulation of synaptic inputs to the neurons also reduced the I(AHP), and had no effect on I(h) or I(A). Phorbol dibutyrate also reduced a background outward current that was present after the I(AHP) current had been blocked by clotrimazole. Both phorbol dibutyrate and ingenol caused translocation of the novel PKC, PKCepsilon, in these neurons. Only phorbol dibutyrate caused translocation of PKCgamma, a conventional PKC. The studies thus indicate that the activation of PKC by phorbol esters and by nerve stimulation affects AH neurons in a similar way, and that PKC activation targets both the I(AHP) and another background K(+) current. The I(AHP) is targeted by a conventional PKC, suggested to be PKCgamma, as this is the only conventional PKC that is prominent in AH neurons.     

Protein kinase C-dependent and -independent effects of phorbol esters on hippocampal calcium channel current

Despite their widespread use in investigations of protein kinase C (PKC), concern is often expressed regarding the specificity of action of phorbol esters. We have extensively compared the effects of PDBu, a phorbol ester that activates PKC, with those of its inactive analog, 4 alpha-PDBu, on calcium (Ca) channel regulation in acutely isolated guinea pig hippocampal neurons and found that PKC-dependent and -independent actions could be clearly distinguished. While both phorbol esters depressed whole-cell barium current through Ca channels (IBa), PDBu was approximately 100-fold more potent than 4 alpha-PDBu. PKC-independent effects began to appear in the range of 5-10 microM, doses that, while high, have been used in some investigations. Moreover, only PDBu (1) was active when applied intracellularly, (2) had effects that were blocked by the PKC inhibitor H-7, and (3) induced PKC translocation with potency similar to its potency in depressing IBa. The finding that 4 alpha-PDBu acted only extracellularly was unexpected and suggested either that it acted via an extracellular binding site or that its orientation in the membrane was crucial to its effects on Ca channels. Finally, (4) PDBu alone caused a hyperpolarizing shift in the voltage dependence of the high-voltage-activated, rapidly inactivating (N type) component of Ca current. This result extends our previous finding that the N-type current component was depressed by PDBu to a greater extent than the L-type component and may represent an important new mode of neurotransmitter regulation of ion channels in the brain via PKC.     

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

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.     

Cross-communication between acetylcholine and VIP in controlling catecholamine secretion by affecting cAMP, inositol triphosphate, protein kinase C, and calcium in rat adrenal medulla

The purpose of the present study was to determine the molecular mechanism of stimulatory actions of ACh and vasoactive intestinal polypeptide (VIP) by determining the role of various second messengers in the neurohumoral secretion. Toward such a goal, we measured cAMP, cGMP, protein kinase (PKC) activity, 3H-inositol triphosphate (3H-IP3), and 45Ca uptake in the adrenal medulla subjected to various treatments. Stimulation of splanchnic nerve endings increased 45Ca uptake, cAMP content, 3H-IP3, and PKC activity in the adrenal medulla. If muscarinic receptors of chromaffin cells were selectively activated by perfusion with muscarine, 3H-IP3 content and PKC activity were enhanced. Nicotine, on the other hand, increased only 45Ca uptake without affecting any other second messenger. Perfusion with VIP increased PKC activity and cAMP and 3H-IP3 content. None of the procedures affected cGMP content. Interplay among various second messengers was further investigated by studying interactions of nicotinic, muscarinic, and VIP-ergic receptors in modulation of catecholamine (CA) secretion and by using agents known to activate specific second messengers (e.g., forskolin, phorbol esters). Our results show that muscarine, VIP, and phorbol ester facilitated nicotine-evoked secretion by increasing PKC activity, and it was associated with an additional increase in 45Ca accumulation. On the other hand, secretion evoked by nicotine as well as muscarine was facilitated by forskolin without additional increase in 45Ca accumulation. A novel feature of the study is that ACh and VIP activate three types of receptors on chromaffin cells to stimulate and mutually facilitate the secretion of CA by generating various second messengers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cyclic AMP regulates processing of neuropeptide precursor in bag cell neurons ofAplysia

The stimulation of a prolonged afterdischarge of action potentials in the bag cell neurons ofAplysia is accompanied by an elevation of cAMP levels in these cells. Such a discharge causes the release of egg-laying hormone (ELH) and several other neuroactive peptides, which are derived from a 32-kDa protein prohormone. We have examined the relationship between the elevation of cAMP levels and the processing of the 32-kDa ELH prohormone. The ELH prohormone was radiolabeled in bag cell clusters by incubation of abdominal ganglia in [³H]leucine and identified on SDS-PAGE by its specific localization to bag cell neurons and its immunoreactivity with antisera to ELH. After labeling the prohormone, further incorporation of [³H]leucine was blocked using either the protein synthesis inhibitor anisomycin or an excess of unlabeled leucine. The stimulation of an afterdischarge, or treatment of cells with the adenylate cyclase activator forskolin or a membrane permeant cAMP analog, resulted in the loss of radiolabeled 32-kDa ELH prohormone relative to that in control clusters. In the presence of tetrodotoxin (TTX), which prevents discharges and stimulation-evoked secretion in the bag cell neurons, forskolin also caused the depletion of labeled ELH prohormone, suggesting that secretionper se is not likely to be required for this effect. The decrease in intensity of the 32-kDa band was accompanied by an increase in a 29-kDa band within the somata. Occasionally, an increase in a group of faint bands with approximateM _r of 26-kDa was observed. Comparative peptide mapping indicated that the 29-kDa protein is likely to be derived from the 32-kDa ELH prohormone. Our findings suggest that elevations of cAMP accelerate and possibly alter the pattern of, processing of the 32-kDa ELH prohormone.     

Activators of adenylate cyclase and cyclic AMP prolong calcium-dependent action potentials of mouse sensory neurons in culture by reducing a voltage-dependent potassium conductance

The effects of compounds that activate adenylate cyclase and of cAMP on calcium-dependent action potentials recorded from mouse dorsal root ganglion neurons were assessed. Application of compounds that stimulate the adenylate cyclase system (forskolin, cholera toxin, and prostaglandin E1) increased action potential duration with an associated decrease in afterhyperpolarization. An adenylate cyclase inhibitor, 2',5'-dideoxyadenosine, partially inhibited the responses to forskolin and cholera toxin. cAMP analogs mimicked the effect of forskolin, and the phosphodiesterase inhibitor theophylline enhanced the response to forskolin. Following intracellular injection of the potassium channel blocker cesium, the forskolin response was reduced. Forskolin did not significantly alter resting membrane potential or conductance. The action potential responses to forskolin were voltage dependent, being reduced when the membrane was held at less negative (less than -50 mV) potentials. The data suggest that activators of adenylate cyclase and cAMP prolong calcium-dependent action potentials by blocking a voltage-dependent potassium conductance that is responsible, in part, for action potential repolarization and that inactivates at membrane potentials less negative than -50 mV.     

Multiple effects of phorbol esters in the rat spinal dorsal horn

Spinal cord slice preparation and intracellular recording techniques were used to examine the effects of phorbol esters on the sodium- and calcium-dependent action potentials, the excitatory synaptic transmission, the basal (resting) and the dorsal root stimulation-evoked release of 9 endogenous amino acids, including glutamate and aspartate, and the responsiveness of the rat dorsal horn neurons to excitatory amino acids (glutamic, kainic, quisqualic, and N-methyl-D-aspartic). 4-beta-Phorbol-12, 13-dibutyrate and 4-beta-phorbol-12, 13-diacetate produced minor alterations in membrane potential and resistance, but they broadened the sodium-dependent action potential and reduced the duration of the calcium-dependent action potential. In addition, phorbol esters caused a marked and long-lasting increase in the amplitude and the duration of excitatory postsynaptic potentials (EPSPs) evoked in dorsal horn neurons by orthodromic stimulation of a lumbar dorsal root. Phorbol esters produced a brief increase in the basal and electrically evoked release of endogenous excitatory (glutamic, aspartic) and inhibitory amino acids (glycine, GABA). In addition, the rates of release of alanine, serine, and threonine were also elevated. In the presence of TTX, phorbol esters selectively enhanced, in a reversible manner, the depolarizing responses of dorsal horn neurons to N-methyl-D-aspartic acid and L-glutamate but not the responses to kainic or quisqualic acids. The potentiation of the NMDA response was blocked by APV, a specific NMDA receptor antagonist. Thus, phorbol esters appear to enhance excitatory synaptic transmission in the rat spinal dorsal horn slice preparation by acting both at pre- and postsynaptic sites. Phorbol esters could potentiate excitatory synaptic transmission by acting predominantly at a postsynaptic site (NMDA receptor), since the duration of the increased responsiveness of dorsal horn neurons to glutamate and NMDA correlates better with the enhancement of EPSPs than with the increased release of the stimulation-evoked glutamate and aspartate. The increased release of endogenous amino acids is consistent with a presynaptic (terminal) site of action, but it could also be explained by enhanced interneuronal activity. Although our results suggest that in the rat spinal dorsal horn protein kinase C may have a role in controlling the release of putative excitatory and inhibitory neurotransmitters and may also be involved in the regulation of postsynaptic NMDA receptors, the identity of endogenous substance(s) participating in these effects is presently unknown.     

Calcium inflow-dependent protein kinase C activity is involved in the modulation of transmitter release in the neuromuscular junction of the adult rat

Using intracellular recording, we studied how protein kinase C activity affected miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in the neuromuscular junctions of the levator auris longus muscle from adult rats. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA, 10 nM) increased the quantal content by approximately 150% (P<0.05). On the other hand, the quantal content decreased by approximately 40% (P<0.05) for all the protein kinase C inhibitors tested (Calphostin-C, 10 microM; Chelerythrine, 1 microM; Staurosporine, 200 nM). These changes in acetylcholine release were maintained at plateau for 1 to 7 h. Moreover, none of the protein kinase C activators or inhibitors used could modify the spontaneous MEPP mean size (P>0.05). We reduced the calcium influx in nerve terminals using the P/Q-type channel blocker omega-Aga-IVA(100 nM) or with 5 mM magnesium in physiological solution. In neither situation was the quantal content modified by PMA or by CaC. However, when high Ca2+ (5 mM) was added to a preparation that was previously blocked with omega-Aga-IVA, PMA and CaC had their full effect. We conclude that under physiological conditions PKC is dependent on the calcium inflow through the P/Q-type voltage-dependent calcium channels during evoked activity and works near the maximum rate at normal external calcium concentration.     

Serotonergic modulation of persistent sodium currents and membrane excitability via cyclic AMP-protein kinase A cascade in mesencephalic V neurons

In rat mesencephalic trigeminal (Mes V) neurons, persistent sodium currents in conjunction with low-threshold potassium currents are critical for generation of subthreshold membrane oscillations and onset of burst behavior. Here we demonstrate that the cAMP/protein kinase A (PKA) signaling pathway modulates persistent sodium currents. In particular, we show that elevation of cAMP suppresses a low-threshold I(NaP) via a PKA intracellular pathway. Bath application of forskolin (20 microM), a stimulant for the production of cAMP, reduced the peak I(NaP). 1,9-Dideoxy-forskolin (20 microM), an inactive form of forskolin, produced minimal effects on I(NaP), and the membrane-permeable cAMP analogue 8-bromo-cAMP (500 microM) mimicked the effect of forskolin. Additionally, preapplication of H89 (2 microM), a specific PKA inhibitor, suppressed the effect of forskolin, suggesting the involvement of the cAMP/PKA intracellular signaling pathway in this modulation. 5-HT receptor stimulation (20 microM) also mimicked the modulation of I(NaP) by forskolin via the cAMP/PKA-dependent signaling pathway. Current clamp analysis demonstrated that voltage-dependent membrane resonance in response to a ZAP input current at depolarized holding potentials (approximately -50 mV) was specifically suppressed by forskolin or 5-HT. Moreover, drug application enhanced frequency adaptation in response to a 1-sec current pulse. These results indicate that modulation of persistent sodium currents by a cAMP/PKA pathway can significantly alter the membrane excitability and discharge characteristics of Mes V neurons.