Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.     

Recovery of hippocampal cholinergic activity by transplantation of septal neurons in AF64A treated rats

Embryonic septal neurons were transplanted into the hippocampus of adult rats which had received lateral-ventricular administration of AF64A, a cholinergic neurotoxin, and the effects on hippocampal cholinergic activity were studied. One week after AF64A administration, we injected dissociated septal cell suspension into the dorsal hippocampus, unilaterally. About 3 months after the transplantation, acetylcholine (ACh)-rich septal grafts formed extensive acetylcholinesterase (AChE)-positive fibers into the host hippocampus, recovering choline acetyltransferase (ChAT) level only in the grafted side. These results indicate that septal implants can produce a partial recovery of the cholinergic activity in the chemically damaged hippocampus.     

γ-干扰素对大鼠胚胎基底前脑及隔区核团胆碱能神经元分化的影响

为了研究γ-干扰素(IFNγ)对大鼠胚胎基底前脑及隔区核团胆碱能神经元分化的作用,采用免疫组织化学方法对胆碱能神经元的特异性标记酶-胆碱乙酰基转移酶(ChAT)进行染色,ChAT阳性细胞的数量反映了胆碱能神经元的数量,并用14C-乙酰CoA作底物来检测ChAT活性。结果显示,IFNγ处理过的实验组,ChAT阳性细胞数量显著增加,ChAT活性也增加,这种增加被大鼠抗小鼠IFNγ单克隆抗体(Ab-IFNγ)完全拮抗。采用流式细胞术对细胞周期进行分析,细胞周期及细胞百分率无明显改变。用MAP2标记神经细胞,神经细胞数基本未增加。以上结果提示:IFNγ不能促进基底前脑和隔区神经元增殖,胆碱能神经元表达增加不是因为神经元数目增加而是分化的结果。     

Insulin-like growth factor-I inhibits endogenous acetylcholine release from the rat hippocampal formation: possible involvement of GABA in mediating the effects

Evidence suggests that insulin-like growth factor-I (IGF-I) plays an important role during brain development and in the maintenance of normal as well as activity-dependent functioning of the adult brain. Apart from its trophic effects, IGF-I has also been implicated in the regulation of brain neurotransmitter release thus indicating a neuromodulatory role for this growth factor in the central nervous system. Using in vitro slice preparations, we have earlier reported that IGF-I potently inhibits K(+)-evoked endogenous acetylcholine (ACh) release from the adult rat hippocampus and cortex but not from the striatum. The effects of IGF-I on hippocampal ACh release was sensitive to the Na(+) channel blocker tetrodotoxin, suggesting that IGF-I might act indirectly via the release of other transmitters/modulators. In the present study, we have characterized the possible involvement of GABA in IGF-I-mediated inhibition of ACh release and measured the effects of this growth factor on choline acetyltransferase (ChAT) activity and high-affinity choline uptake in the hippocampus of the adult rat brain. Prototypical agonists of GABA(A) and GABA(B) receptors (i.e. 10 microM muscimol and 10 microM baclofen) inhibited, whereas the antagonists of the respective receptors (i.e. 10 microM bicuculline and 10 microM phaclofen) potentiated K(+)-evoked ACh release from rat hippocampal slices. IGF-I (10 nM) inhibited K(+)- as well as veratridine-evoked ACh release from rat hippocampal slices and the effect is possibly mediated via the activation of a typical IGF-I receptor and the subsequent phosphorylation of the insulin receptor substrate-1 (IRS-1). The inhibitory effects of IGF-I on hippocampal ACh release were not additive to those of either muscimol or baclofen, but were attenuated by GABA antagonists, bicuculline and phaclofen. Additionally, in contrast to ACh release, IGF-I did not alter either the activity of the enzyme ChAT or the uptake of choline in the hippocampus.These results, taken together, indicate that IGF-I, under acute conditions, can decrease hippocampal ACh release by acting on the typical IGF-I/IRS receptor complex while having no direct effect on ChAT activity or the uptake of choline. Furthermore, the evidence that effects of IGF-I could be modulated, at least in part, by GABA antagonists suggest that the release of GABA and the activation of its receptors may possibly be involved in mediating the inhibitory effects of IGF-I on hippocampal ACh release.     

Does the release of acetylcholine in septal slices originate from intrinsic cholinergic neurons bearing p75(NTR) receptors? A study using 192 IgG-saporin lesions in rats

In previous studies electrically-evoked release of acetylcholine in septal slices was demonstrated. The present experiment aimed at verifying if this release involved intrinsic neurons bearing p75(NTR) receptors. Long-Evans rats sustained injections of 192 IgG-saporin into the medial septum/diagonal band of Broca (0.8 microg). Sham-operated rats served as controls. Two to 3.5 weeks later, the electrically-evoked release of acetylcholine ([(3)H]ACh) was measured in slices from the lateral septum (LS), medial septum (MS) and diagonal band of Broca (DBB). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and monoamine concentrations were measured in the septum, cortex and hippocampus. The lesion extent was also assessed by ChAT immunostaining in a separate series of rats. In the septum, the number of ChAT-positive neurons was depleted dramatically (>90% at the level of the injection site). In the hippocampus, the lesions reduced ChAT and AChE activity by 91% and 84%, respectively. In the cortex, this reduction was weaker (-55% and -47%). In the septal region, the reduction was either weak or not significant. The evoked release of acetylcholine in septal slices was not reduced, except in the slices from the LS (-64%). The effects of physostigmine and atropine confirmed the presence of autoreceptors. Our data exclude that a major part of the acetylcholine released by MS and DBB slices derived from intrinsic neurons bearing p75(NTR) receptors. In the LS, part of the released acetylcholine might be from projections of such neurons located in the LS, MS and/or DBB. These data also suggest that the MS and the DBB may be the target of extrinsic cholinergic innervation that does not bear p75(NTR) receptors.     

Basic fibroblast growth factor suppressed the enhancement of choline acetyltransferase activity induced by nerve growth factor

We investigated change of choline acetyltransferase (ChAT) activities in rat embryonic (16 days) septal neuron culture in treatment with basic fibroblast growth factor (bFGF) and nerve growth factor (NGF). Total ChAT activity increased by addition of bFGF, NGF, and bFGF plus NGF with dose-dependent manner. NGF showed much enhancement of specific ChAT activities per mg protein, but bFGF or bFGF plus NGF, respectively showed little or slightly enhanced ChAT activities. In histochemical studies with anti-ChAT antibody staining, cholinergic neurons in NGF-treated culture were stained more strongly than those in other conditioned cultures such as control, bFGF-treated, and bFGF plus NGF-treated cultures. These results suggest that bFGF enhances total ChAT activity but not cellular ChAT activity and further suppresses the enhancement of cellular ChAT activity induced by NGF in septal neuron culture.     

Effect of unilateral decortication on acetylcholine release from the nucleus basalis

Acetylcholine (ACh) is released from the rat nucleus basalis magnocellularis (nbM) following tissue depolarization with 35 mM K+. In the present study, we report that cholinergic neurons within the nbM undergo retrograde changes resulting in a significant reduction in K+-evoked ACh release following cortical lesions.     

Long term effects of septohippocampal lesions and intrahippocampal grafts on acetylcholine concentration,muscarinic stimulated formation of inositol phospholipids and electrically evoked release of neurotransmitters in the rat hippocampus

Summary Long Evans female rats sustained aspirative lesions of the septohippocampal pathways; subsequently, they received intrahippocampal suspension grafts of fetal septal-diagonal band or hippocampal tissue. The long term (8–10 months post-surgery) effects of these treatments were examined in the hippocampus for the following variables: concentration of hippocampal acetylcholine (ACh), muscarinic-stimulated (carbachol) formation of inositol monophosphate, accumulation of tritiated choline, noradrenaline (³H-NA) and serotonin (³H-5-HT), electrically evoked release of ³H-acetylcholine (³H-ACh), ³H-NA and ³H-5-HT, and choline acetyltransferase (ChAT) activity. The lesions decreased the levels of endogeneous ACh, the accumulation of ³H-choline and ³H-5-HT and the evoked release of both ³H-ACh and ³H-5-HT as well as the ChAT activity, but they failed to significantly affect the muscarinic-stimulated formation of inositol monophosphate and the accumulation and release of ³H-NA. Grafts of hippocampal cells were found to be ineffective on all lesion-induced effects. In contrast, grafts of septal-diagonal band origin attenuated the deficit of hippocampal concentrations of ACh and accumulation of ³H-choline without, however, improving release of ³H-ACh, accumulation and release of ³H-5-HT, and ChAT activity. These observations suggest that: (i) denervation-induced hippocampal muscarinic supersensitivity might not be long-lasting or the lesions, which in some cases spared the lateral edges of the fimbria, failed to induce any muscarinic supersensitivity, (ii) intrahippocampal grafts rich in cholinergic neurons do not foster recovery from the lesion-induced noncholinergic deficits we assessed, (iii) recovery of function may be expressed by some but not all biochemical or pharmacological cholinergic variables and (iv) graft-derived hippocampal reinnervation may be less efficient than the endogenous innervation of intact rats as indicated by the restoration of only some of the variables related to cholinergic function by intrahippocampal septal-diagonal band grafts.     

Increase in chloride permeability of snail neurons during high potassium-induced hyperpolarization

High K+-induced hyperpolarization was recorded intracellularly from the snail neurons, Euhadra subnimbosa, and the ionic mechanism underlying this hyperpolarization was analyzed in comparison with the ACh-induced hyperpolarization of the same cell, the latter known to be Cl(-)-dependent. The membrane resistance always decreased during both hyperpolarizing responses to high K+ (24mM) and ACh (0.1 mM). Both hyperpolarizing responses to high K+ and ACh were reversed in Cl(-)-free Ringer to the depolarizing responses. Both hyperpolarizing responses to high K+ and ACh were markedly augmented immediately after returning to normal Cl- from Cl(-)-free Ringer perfusion. Increase in intracellular Cl(-)-concentration by a leak from KCl-electrode reversed both hyperpolarizing responses to high K+ and ACh. Reversal potential of high K+-response was always 10-20 mV more positive than that of ACh-response, when measured in normal Ringer perfusion. Intracellular Cl(-)-concentration of the cells which were hyperpolarized by high K+ was estimated to be one half of that of the cells which were depolarized by high K+. Above results indicated that the high K+-induced hyperpolarization is due to the permeability increase of the postsynaptic membrane toward Cl-, masking the depolarizing effect of high K+ on the same membrane.     

Stereological estimates of the basal forebrain cell population in the rat, including neurons containing choline acetyltransferase, glutamic acid decarboxylase or phosphate-activated glutaminase and colocalizing vesicular glutamate transporters

The basal forebrain (BF) plays an important role in modulating cortical activity and influencing attention, learning and memory. These activities are fulfilled importantly yet not entirely by cholinergic neurons. Noncholinergic neurons also contribute and comprise GABAergic neurons and other possibly glutamatergic neurons. The aim of the present study was to estimate the total number of cells in the BF of the rat and the proportions of that total represented by cholinergic, GABAergic and glutamatergic neurons. For this purpose, cells were counted using unbiased stereological methods within the medial septum, diagonal band, magnocellular preoptic nucleus, substantia innominata and globus pallidus in sections stained for Nissl substance and/or the neurotransmitter enzymes, choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD) or phosphate-activated glutaminase (PAG). In Nissl-stained sections, the total number of neurons in the BF was estimated as approximately 355,000 and the numbers of ChAT-immuno-positive (+) as approximately 22,000, GAD+ approximately 119,000 and PAG+ approximately 316,000, corresponding to approximately 5%, approximately 35% and approximately 90% of the total. Thus, of the large population of BF neurons, only a small proportion has the capacity to synthesize acetylcholine (ACh), one third to synthesize GABA and the vast majority to synthesize glutamate (Glu). Moreover, through the presence of PAG, a proportion of ACh- and GABA-synthesizing neurons also has the capacity to synthesize Glu. In sections dual fluorescent immunostained for vesicular transporters, vesicular glutamate transporter (VGluT) 3 and not VGluT2 was present in the cell bodies of most PAG+ and ChAT+ and half the GAD+ cells. Given previous results showing that VGluT2 and not VGluT3 was present in BF axon terminals and not colocalized with VAChT or VGAT, we conclude that the BF cell population influences cortical and subcortical regions through neurons which release ACh, GABA or Glu from their terminals but which in part can also synthesize and release Glu from their soma or dendrites.     

银杏叶提取物改善AD模型鼠学习记忆功能的作用

目的 :研究银杏叶提取物 (EGb)对早老性痴呆 (AD)模型鼠学习记忆等认知功能的影响和对基底前脑隔区胆碱能神经元的保护作用。方法 :大鼠随机分成预防组、治疗组和对照组。各组切割右侧海马伞后分别用EGb(预防组及治疗组 )和蒸馏水 (对照组 )灌胃连续 6周。其中预防组在手术前先用EGb灌胃 2周。各组在术后次日及灌胃 6周后进行行为学检测。脑切片经Nissl、NOS组织化学和ChAT免疫组织化学染色 ,观察损伤侧内侧隔核和斜角带垂直支中NOS和ChAT阳性神经元的变化 ,并对ChAT阳性神经元作图像分析。数据分别用方差分析、SNK检验及 χ2 检验作统计学处理。结果 :行为学检测证实 ,EGb用药组AD模式鼠的学习记忆等认知功能较对照组明显改善 ,其中预防组效果又较治疗组为好 ;形态学检测证实 ,EGb用药组AD模式鼠损伤侧内侧隔核和斜角带垂直支中NOS、ChAT阳性神经元数目及ChAT阳性神经元的面积和周径等均优于对照组 ,预防组结果又优于治疗组。结论 :EGb对AD模型鼠学习记忆等认知功能障碍有一定的改善作用 ,这一作用是通过保护基底前脑隔区胆碱能神经元而实现的 ,其中预防效果比治疗效果更优     

3H]acetylcholine release and the change in cytosolic free calcium level induced by high K+ and ouabain in rat brain synaptosomes

High K+ (50 mM) increased both [3H]acetylcholine ([3H]ACh) release and cytosolic free calcium level ([Ca2+]i) in rat brain synaptosomes in the presence of extracellular Ca2+. Ouabain (5 x 10(-8) to 5 x 10(-4) M) also caused a dose-dependent increase in [3H]ACh release, but not in [Ca2+]i, in the absence of Ca2+. The effects of high K+ and ouabain on [3H]ACh and/or [Ca2+]i, were inhibited by the intracellular Ca2+ antagonist TMB-8 (10(-4) M). These results suggest that unlike high K+, ouabain increases transmitter release from nerve endings through a mechanism which is independent of [Ca2+]i, but sensitive to TMB-8.     

Cholinergic properties of embryonic chick sensory neurons

Experiments were carried out to determine the cholinergic properties of sensory neurons of the chick embryo by measuring the choline acetyltransferase activity (ChAT) and [3H]choline uptake. The choline acetyltransferase activity in the dorsal root ganglia of an 8-day-old chick embryo was 24.2 +/- 2.52, which increased to 45.4 +/- 9.69 pmol ACh/mg protein/min in the ganglia of 12-day-old embryos. Sensory neurons derived from dorsal root ganglia of 10-day-old embryos and maintained in a serum-free culture medium supplemented with insulin, transferrin and nerve growth factor (NGF) also contained significant amounts of ChAT (21.9 pmol ACh/mg protein/min). Omission of NGF resulted in neuronal death, and the enzyme activity could not be measured in these cultures. A specific inhibitor of ChAT, hydroxyethyl naphthylvinyl pyridine (NVP), when added to the assay mix produced a dose-dependent inhibition of ChAT from cultured neurons. Cultured sensory neurons incubated with [3H]choline followed by repeated washouts took up and retained [3H]choline. The uptake of [3H]choline was reduced by about 45% when NaCl, in the incubation medium, was replaced by LiCl. A specific inhibitor of choline uptake, hemicholinium-3, caused about 75% inhibition of [3H]choline uptake. It is implied that sensory neurons of the chick dorsal root ganglia express cholinergic properties during development.     

K(+)- and temperature-evoked taurine efflux from hypothalamic astrocytes

Hypothalamic astrocytes in culture released taurine, a suspected inhibitory amino acid neurotransmitter/neuromodulator/osmoregulator, in response to isoosmotically increasing extracellular K+ in a dose-dependent fashion. In the absence of added Ca2+, basal release levels rose to approach those obtained after exposure to 60 mM K+ in the presence of 2.5 mM Ca2+, and were only partially lowered by the addition of 10 mM Mg2+. Stimulation with K+ (60 mM) did not further increase taurine efflux above the high basal levels seen in the absence of Ca2+. Under standard conditions complete replacement of Na+ with choline Cl had little effect on basal taurine release, but reduced K(+)-evoked (60 mM) efflux by 60%. The temperature dependence of the basal levels of taurine released from hypothalamic astrocytes was similar to that seen for cultured cerebellar astrocytes and neurons over the range 5-50 degrees C. Taurine release increased from 5 to 15 degrees C, remained constant between 15 and 33 degrees C, decreased between 33 and 37 degrees C and increased thereafter. The infection point of increased basal taurine release seen around 37 degrees C (most prominent in astrocytes), may be of physiological significance. Results presented also show that the ion (Na+, Ca2+ and K+) sensitivities of taurine efflux for cultured hypothalamic astrocytes are similar to those previously reported for cultured astrocytes from the cerebellum.     

GABA antagonists increase the release of [3H]acetylcholine from the chick retina

The release of [3H]acetylcholine from the chick retina was studied. A 5 mM increase in K+-concentration caused an increased release, which was Ca2+-dependent. The effect of 5 mM K+ was neither potentiated by bicuculline nor inhibited by isoguvacine or muscimol. This indicates that the K+-induced release is not controlled by GABA. However, bicuculline and picrotoxin increased the spontaneous efflux of radioactivity, whereas GABA had no significant effect. The results suggest that cholinergic neurons are tonically inhibited by a continuous release of endogenous GABA. Neither glycine or strychnine, nor dopamine or haloperidol had any effect on the spontaneous release.     

Calcium-activated potassium channels recorded from rat neocortical neurons in cell culture

Rat neocortical neurons in cell culture were studied with the patch-clamp technique in order to determine the properties of a large-conductance K+ channel in excised inside-out patches. In the presence of a physiological ionic gradient for K+ across the patch membrane ([K+]i = 120 mM; [K+]o = 3 mM), outward channel activity was detected when the patches were brought to membrane potential values less negative than -30 mV. Depolarization of the membrane increased the magnitude of the current. The I-V relationship displayed rectification at negative membrane potentials. When the I-V curve was differentiated the slope conductance calculated at 0 mV membrane potential was 120 pS. The single-channel permeability was 5.2 x 10(-13) cm/s and the current flow through the open K+ channel could be modeled using the constant-field electrodiffusion theory. K+ channel opening was not observed following removal of Ca2+ from the intracellular surface of the membrane. Our experiments indicate that, as in other cell types, rat neocortical neurons in culture exhibit a large-conductance K+ channel which is activated by Ca2+ acting on the cytoplasmic surface.     

Termination of transmitter release by stimulation of sodium-potassium activated ATPase.

1. The release of acetylcholine (ACh) from Auerbach's plexus of guinea-pig ileum has been measured in eserinized Krebs solution using longitudinal muscle strip preparations. 2. Removal of the external K ions enhanced both the resting and stimulated release of ACh from the plexus. This effect was not affected by tetrodotoxin. 3. On readmission of K+ to tissues which had been suspended in K-free Krebs solution the release of ACh was promptly reduced in both stimulated and unstimulated tissues. The extent of the reduction of ACh release depended on the exposure time to K-free solution, the recovery being delayed by longer exposure. 4. The ACh releasing effect of (1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP) was completely inhibited by the readmission of K ions to tissue which had been kept in K-free Krebs solution. 5. Rb+ substitution for K+ produced no change in ACh release and addition of 5-9 mM-Rb after K removal reduced the release of ACh as K did readmission. When the K ions were substituted by Cs+, both the resting and stimulated release were enhanced. The amount of ACh released by a stimulus was enhanced both at low and high frequency of sustained stimulation. 6. Removal of the external K ions increased the release of tritiated noradrenaline (NA), from isolated rat iris; however, when K+ (5-9 mM) was readmitted the release was reduced even below the control value. 7. It is concluded that the stimulation of (Na+-K+)-activated ATP-ase in the membrane inhibits the release of transmitter, and under physiological condition Ca-fluxes and the subsequent inhibition of membrane ATP-ase may be involved in triggering the release of transmitter.     

The effects of K+ channel blockers on ovine parotid secretion depend on the mode of stimulation

The vascularly isolated parotid glands of sheep were submaximally stimulated for 4 min by nerve stimulation, or by infusion of acetylcholine (ACh) or bethanechol directly into the artery supplying the gland. The three modes of stimulation caused almost equal increases in the rate of salivary flow, initial losses of K+ and phosphate from the gland and total glandular deficits of K+ and phosphate. Concurrent arterial infusion of the K+ channel blocker tetraethylammonium (TEA), at 1.5-3.3 mM in blood, almost abolished these responses to bethanechol but did not alter the responses to nerve stimulation or ACh. The responses to bethanechol were restored by increasing the concentration fourfold. Concurrent arterial infusion of 4-aminopyridine (4-AP), at 0.1-2.3 mM in blood, partially inhibited the increase in salivary flow due to bethanechol but not the response to nerve stimulation or ACh. The specificity and competitive nature of the action of TEA and 4-AP on responses to direct muscarinic stimulation by bethanechol are consistent with blockade of K+ channels in secretory end-piece cells. The failure of TEA and 4-AP to inhibit responses to nerve stimulation and ACh may be due to the blocking agents potentiating the release of neurotransmitter ACh. It is also possible that nerve stimulation and ACh may cause the release of a co-transmitter which bethanechol does not.     

Effects of pertussis toxin on the affinity of exocytosis for Ca2+ in bovine adrenal chromaffin cells

Effects of pertussis toxin (islet-activating protein, IAP) on the secretory function of bovine adrenal chromaffin cells in culture were studied. Treatment of chromaffin cells with IAP resulted in an increase in both basal release of catecholamine and evoked-release by either acetylcholine (ACh) or high K+. In the dose-response curve for ACh-evoked release, IAP treatment produced an increase of the maximal response without affecting the half-maximal concentration of ACh. When the cells were permeabilized with digitonin after IAP-pretreatment, Ca2(+)-dependent exocytosis was markedly increased where the affinity of exocytosis for Ca2+ was augmented. These findings suggest that IAP-sensitive GTP-binding protein (or proteins) directory controls the Ca2(+)-triggered process in the machinery of exocytosis by modulating the affinity for Ca2+ of its unknown target.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.