Enkephalin is a competitive antagonist of cholecystokinin in the gastrointestinal tract, as predicted from prior conformational analysis

Prior calculations based on ECEPP (Empirical Conformational Energies for Peptides Program) of the low energy minima for cholecystokinin (CCK) and Met-enkephalin have demonstrated that significant structural features of these two peptides are identical. This result suggested the possibility that Met-enkephalin, as well as other enkephalin analogues of similar structure, could associate with receptors for CCK. To test this theoretical result, we examined the ability of Met-enkephalin and its analogues to bind to peripheral CCK receptors in the rat gastrointestinal tract; in particular, we measured the ability of the opiate peptide to inhibit the effects of CCK in a physiological assay system which we have previously characterized: CCK-induced contraction of the isolated rat pyloric sphincter. We find that Met-enkephalin is an antagonist of the CCK-8-induced contraction, with a IC₅₀ of 110 nM. Furthermore, antibodies against CCK were found to cross-react with Met-enkephalin and its analogues in a manner which suggests a distinct structure-activity relationship. These experimental results strongly support the theoretical results of conformational analysis showing structural similarity between enkephalin and CCK. They further suggest that enkephalins could modulate the response of CCK systems under physiological conditions.     

Preparation of an N-acetyl-octapeptide of cholecystokinin: The role of N-acetylation in protecting the octapeptide from degradation by smooth muscle tissues

The C-terminal octapeptide of cholecystokinin (CCK-8) was acetylated on its lone N-terminal amino group using acetic anhydride in N,N-dimethylformamide. The acetylated derivative (Ac-CCK-8) and unreacted CCK-8 were separated from acetic anhydride and other reaction products by fractionation on Sephadex LH-20. Final purification was by thin-layer isoelectric focusing in a pH 2.5–4.0 gradient. The immunochemical properties of the octapeptide were unaffected by acetylation as measured by radioimmunoassay. The N-acetylated-octapeptide was equally as effective as unmodified CCK-8 in producing concentratiion-dependen isometric tension development in isolated cat gallbladder strips. Acetylation did, however, protect CCK-8 from N-terminal degradation by soluble peptidases that eluted from gallbladder and other smooth muscle tissues of the cat. Unmodified CCK-8 was degraded rapidly in the presence of these tissues and in buffers previously exposed to the same tissues. In contrast, the Ac-CCK-8 was resistant to N-terminal degradation under the same conditions. Degradation of CCK-8 from its N-terminus produces biologically inactive derivatives and could adversely affect in vitro studies. Since the acetylated-CCK-8 retained full biological and immunological activity, its use would eliminate the effect of extracellular proteolysis on CCK-8 action.     

CCK-8对内毒素休克大鼠肺脏细胞因子的抑制效应

观察八肽胆囊收缩素(cholecystokinin-octapeptide,CCK-8）改善脂多糖(lipopolysaccharide,LPS）引起的大鼠内毒素性休克（endotoxic shock,ES）过程中血清及肺脏细胞因子的变化,探讨p38比裂素活化蛋白激酶（p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入LPS(p38 mito-gen-activated protein kinase,p38 MAPK）的信号转导作用。用生理多道记录仪观察尾静脉注入 LPS（8mg/kg i.v.）复制的SD大鼠ES模型、LPS注入前10min尾静脉注入CCK-8(40ug/kg i.v.）、单独注入CCK-8(40Uug/kg i.v.）或生理盐水（对照）的四组大鼠平均动脉血压（MAP）的改变,应用ELISA试剂盒检测血清和肺脏中炎性细胞因子（TNF-a、IL-1β和IL-6）的变化。用Western blot检测肺脏p38 MAPK的表达。结果显示：CCK-8可改善LPS引起的大鼠MAP的下降。与对照组相比,LPS可显著增加血清和肺脏TNF-a、IL-1β和IL-6含量;CCK-8可显著抑制LPS诱导的血清和肺脏TNF-a、IL-1β和IL-6的增加。CCK-8可增加ES大鼠肺脏磷酸化p38 MAPK的表达。结果提示CCK-8可改善ES大鼠MAP的降低,并对肺脏促炎性细胞因子过量产生有抑制作用,p38MAPK可能参与了其信号转导机制。     

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.     

兔侧脑室注射八肽胆囊收缩素抗血清对血浆自由脂肪酸浓度的影响

本工作根据抗原抗体间具有高度特异性的中和作用的原理,将微量 CCK-8抗血清注射入制备有埋藏套管的慢性实验兔的侧脑室内,观察在中和脑内外源性或内源性的 CCK-8后,血浆 FFA 浓度的变化,结果如下:1.侧脑室内注射正常兔血清,对血浆 FFA 浓度无明显影响;在注射 CCK-8同时注射兔正常血清,也不影响 CCK-8降低血浆 FFA 浓度的作用。2.侧脑室内注射 CCK-8抗血清能有效地阻断外源性脑室注射 CCK-8降低血浆 FFA 的作用,此阻断作用随 CCK-8抗血清剂量的增加而增强。3.侧脑室内单独注射 CCK-8抗血清,血浆 FFA 浓度明显升高,这一作用可能是由于中和了脑内内源性 CCK-8的作用所致。以上结果表明,CCK-8脑室注射引起的血浆 FFA 降低的作用具有一定的特异性;而在正常生理情况下,脑内释放的内源性 CCK-8可能参与血浆 FFA 代谢的调节。     

Alkaline extraction of cholecystokinin-immunoreactivity from rat brain

Alkaline aqueous extractants remove from rat brain 2 to 4 times the CCK-immunoreactivity that is removed by acidic or neutral aqueous extractants. The distribution among the various hormonal forms appears to be independent of the extractant: about $\text{1}\text{10}$ in the larger basic forms (CCK-33 and CCK-39); about $\text{1}\text{4}$ as the C-terminal dodecapeptide (CCK-12) and the remainder as the octapeptide (CCK-8). In contrast, alkaline and acidic aqueous solutions are equally efficient in extraction of enkephalin-immunoreactivity from the same tissues. We are presently unable to account for the very different efficiencies of the various extractants in removing CCK-immunoreactivity from brain.     

八肽胆囊收缩素对吗啡抑制大鼠离体空肠电与收缩活动的对抗作用

本研究探讨了八肽胆囊收缩素（ＣＣＫ－８）对抗吗啡对大鼠离体空肠电与收缩活动的作用。结果表明,吗啡能抑制ＡＣｈ对空肠峰波发放和收缩的加强作用;ＣＣＫ－８可对抗吗啡的作用;在此基础上,ＣＣＫ－Ａ受体拮抗剂ｄｅｖａｚｅｐｉｄｅ（１０ｎｍｏｌ／Ｌ）能完全翻转ＣＣＫ－８的抗吗啡作用,但是ＣＣＫ－Ｂ受体拮抗剂Ｌ－３６５,２６０在１０ｎｍｏｌ／Ｌ时可部分翻转、在３０ｎｍｏｌ／Ｌ时能完全翻转ＣＣＫ－８的作用。上述     

八肽胆囊收缩素对大鼠颈动脉窦压力感受器反射的抑制作用

在36只隔离灌流颈动脉窦区的麻醉大鼠上,观察了八肽胆囊收缩素(cholecystokinin octapepide,CCK-8)对颈动脉窦压力感受器反射的影响。其结果如下：(1)以CCK-8(0．1、0．5、1．0μmol／L)隔离灌流颈动脉窦区时,压力感受器机能曲线向右上方移位,曲线最大斜率(peak slope,PS)减小,反射性血压下降幅度(reflex decrease,RD)减少,阈压(threshold pressure,TP)和饱和压(saturation pressure,SP)均增高。其中RD、PS和TP呈明显的剂量依赖性;(2)用CCK-8的非特异性受体拮抗剂丙谷胺(100μmol／L)预处理后,能明显减弱CCK-8(0．50mol/L)对压力感受器反射的抑制;(3)预先灌流一氧化氮合酶(nitric oxide synthase,NOS)阻断剂(L-NAME,100μmol／L),不能阻断CCK-8(0．5μmol/L)对压力感受器反射的影响;(4)用Ca^2 通道激动剂Bay K 8644(500nmol／L)预处理后,也能明显减弱CCK-8(0．5μmol／L)对压力感受器反射的抑制作用。以上结果提示,CCK-8是通过作用于颈动脉窦压力感受器神经元末梢上的受体而起到抑制作用的,其机制可能为抑制了牵张敏感性通道,致使Ca^2 离子内流减少,而与内皮细胞释放NO无关。     

八肽胆囊收缩素激活钙离子通道和酪氨酸激酶诱导豚鼠心肌细胞内的游离钙增高

探讨八肽胆囊收缩素(CCK-8)对豚鼠单个心肌细胞内游离钙浓度([Ca2+]i的影响及其信号转导机制.Fluo 3-AM标记酶消化法分离的单个心室肌细胞,用激光共聚焦显微镜测定细胞内[Ca2+]i的浓度.[Ca2+]i的变化用荧光强度(Fi)和相对荧光强度(Fi/F0%)表示.实验结果如下(1)在含Ca2+1.0 mmol/L的Tyrode's液中,CCK-8(1～104pmoVL)均可引起[Ca2+]i快速显著上升(P＜0.01).(2)用钙离子鳌合剂EGTA(3 mmol/L)和钙离子通道阻断剂nisoldipine(0.5μmol/L)预孵育心肌细胞5 min,CCK-8(102pmol/L)仅可引起[Ca2+]i缓慢轻度上升(P＜0.01).(3)用非选择性CCK受体拮抗剂丙谷胺(proglumide 6μmo1/L)或酪氨酸激酶抑制剂genistein(1 μmol/L)预孵育心肌细胞5 min,则完全抑制CCK-8诱导的[Ca2+]i升高(P＜0.01).CCK-8可通过激活其受体控制的Ca2+通道,引起Ca2+内流,诱导细胞内Ca2+释放,引起豚鼠单个心肌细胞内[Ca2+]i上升,此作用可能由酪氨酸激酶介导.     

缩胆囊素、胃泌素及丙谷胺对大鼠胆汁分泌的影响

本文旨在研究五肽胃泌素(P-Gas)、缩胆囊素(CCK)与其受体拮抗剂丙谷胺(PGM)单独静脉输注或联合应用对大鼠胆汁流量及胆汁成分排泌量的影响。结果表明:(1)P-Gas无利胆作用;(2)CCK-8仅少量增加胆汁及 HCO_3~-的分泌量;(3)PGM 明显地增加胆汁及胆汁中 HCO_3~-和 CI~-的排泌量,但胆酸分泌量不增加;(4)当 CCK-8 2.3μg/(kg·h)静脉灌注与 PGM 200mg 灌胃联合应用时,胆汁、HCO_3~-和 CI~-的排泌量进一步增加,但P-Gas 2μg/(kg·h)与 PGM 联合应用时的胆汁排泌量低于单独应用 PGM 组。以上结果证明:PGM 有明显的促胆汁分泌作用,其机制属于非胆酸依赖性利胆、而CCK-8只是胆汁分泌的微弱刺激剂,P-Gas 则无利胆作用。在 CCK-8与 PGM 二组间,似有促胆汁分泌的协同作用;但表现在 P-Gas 与 PGM 二组间的作用,似为一种拮抗作用。     

八肽胆囊收缩素对内毒素休克时海马损伤的影响及其机制初探

目的：观察八肽胆囊收缩素（CCK-8）对内毒素休克（ES）时海马损伤的影响,并探讨其可能的作用机制。方法：将日本大耳白兔经静脉注入内毒素的主要活性成分脂多糖（LPS,8mg／kg）复制ES模型。动物（32只）随机分为对照组、LPS组、CCK-8＋LPS组和非特异性CCK受体拮抗剂丙谷胺（Pro）＋LPS组（n=8）。监测平均动脉压（MAP）的变化,光、电镜观察海马的组织形态学改变,比色法检测海马NOS和SOD活性、N0和MDA含量的改变．用SD大鼠（12只,同上复制模型及分组）以免疫组织化学染色法观察海马iNOS和nNOS表达的变化。结果：与对照组相比,注入LPS后出现MAP显著而持续下降（P〈0．01）;海马部位神经元损伤明显;iNOS和nNOS表达增强,NOS活性、NO和MDA含量显著升高（P〈0．05、P〈0．01和P〈0．01）,SOD活性则降低（P〈0．01）。预先注入CCK-8可明显减轻上述变化,预先注入Pro则加剧以上变化。结论：CCK-8可减轻ES时脑内海马部位的损伤。其机制可能与其抗氧化作用和抑制NO的过量生成有关。     

The effects of γ-hexachlorocyclohexane on amylase secretion and inositol phospholipid metabolism in mouse pancreatic acini

Dispersed mouse and guinea-pig pancreatic acini were used to examine the effects of the inositol analogue, γ-hexachlorocyclohexane (lindane) on agonist-stimulated amylase secretion. Secretion from mouse acini in response to carbachol and cholecystokinin octapeptide (CCK-8) was reduced by lindane. Similarly, amylase release from guinea-pig acini stimulated by carbachol was abolished by lindane. These acini, however, still remained responsive to dibutyryl-cAMP with only a slightly diminished secretion to this agent. Inositol phospholipid synthesis and hydrolysis was stimulated in mouse acini by both carbachol and CCK-8. Although hydrolysis of these lipids in response to CCK-8 was reduced by only 18%, stimulation of inositol phospholipid synthesis by either agonist was abolished by lindane. Dose-response curves for inositol phospholipid synthesis stimulated by carbachol and CCK-8 in mouse acini were biphasic and superimposable with those of amylase secretion. In contrast, the dose-response curve for phosphoinositide hydrolysis was sigmoid and clearly separable from that of synthesis. Reducing the external Ca²⁺ concentration caused the dose-response curves for carbachol- and CCK-8-induced inositol phospholipid synthesis to be displaced to the right, as has been observed for amylase secretion. A23187 was also found to induce amylase secretion and inositol phospholipid synthesis, and both of these responses were inhibited by lindane. Amylase secretion and inositol phospholipid synthesis may, therefore, be closely related events in the exocrine pancreas. Lindane may provide a valuable tool with which to determine the role of inositol phospholipid metabolism in stimulus-response coupling.     

八肽胆囊收缩素对大鼠心功能的影响及受体机制

为探讨八肽胆囊收缩素 (CCK 8)对麻醉大鼠心功能的影响及受体机制 ,实验监测了左心室收缩压(LVP)、左心室收缩与舒张期内压变化的最大速率 (±LVdp/dtmax)、心率 (HR)和平均动脉压 (MAP)。结果如下 :小剂量CCK 8(0 4 μg/kg)可引起心动过速 ,MAP、LVP和±LVdp/dtmax轻度上升 ;中剂量CCK 8(4 μg/kg)和大剂量CCK 8(4 0 μg/kg)可引起心动过缓 ,MAP、LVP和±LVdp/dtmax显著增加 ;应用CCK 受体 (CCK R)拮抗剂丙谷胺 (1 0mg/kg)抑制以上变化 ;由逆转录 聚合酶链反应 (RT PCR)检测到心肌组织有CCK A受体 (CCK AR)和CCK B受体 (CCK BR)mRNA表达。以上结果提示 :CCK 8可激活心肌组织的CCK R ,引起剂量依赖性的心功能增加和心率改变。     

Electrophysiological Responses of Nucleus Tractus Solitarius Neurons to CCK and Gastric Distension in Newborn Lambs

The effect of cervical vagus nerve stimulation, gastric distension and CCK-8S administration was studied on the activity of 120 neurons located in the nucleus tractus solitarius (NTS) of anesthetized newborn lambs. One hundred cells responded to the three different inputs.The distribution of the cells in the NTS was from 3 mm rostral to 3 mm caudal to the obex, the major responsive cells being located at the level of the obex. Neurons were either excited or inhibited by gastric distension and CCK-8S, and the responses to these two stimuli were always in the same direction. A small number of cells responded to gastric distension and CCK-8S but not to vagus nerve stimulation.Injection of the CCK-A receptor antagonist 2-NAP abolished both the responses to CCK-8S and to gastric distension. The results are consistent with the idea that CCK-8S acts directly on vagal mechanoreceptive endings in the gastric corpus close to duodenum.These results from lambs may reflect the pathway by which gastric distension and peripheral CCK-8S modulate NTS cells activity during colostrum ingestion, which could in turn activate structures related to learning and memory processes involved in the development of mother preference.     

八肽胆囊收缩素(CCK_8)的人工合成

本文报道了用液相法合成八肽胆囊收缩素(H-Asp-Tyr(SO_3H)-Met-Gly-Trp-Met-Asp-Phe-NH_2,ccK_8)。先分别合成C-端四肽和N-端四肽,然后缩合成八肽,并用温和的乙酰硫酸吡啶盐(PAS)法使其酪氨酸的酚羟基硫酯化。根据其氨基酸组成,层析行为和生物活性等证实产物为纯品。     

Degradation of cholecystokinin octapeptide by the neutral endopeptidase EC 3.4.24.11 and design of proteolysis-resistant analogues of the peptide

Inactivation of cholecystokinin octapeptide in vitro involves a metalloendopeptidase (EC 3.4.24.11) also called enkephalinase that inactivated the peptide both by a sequential pathway of hydrolysis (removal of Phe-NH₂ followed by cleavage of Trp-Met-Asp) and by an endopeptidase action (production of the tetrapeptides).

As enkephalinase cleaved CCK-8 at the Gly⁴-Trp⁵, Trp⁵-Met⁶ and Asp⁷-Phe⁸ bonds, we investigated the stability of analogues having: (1) substitutions of amino acids by a stereoisomer, (2) a substitution of Asp⁷ by a β Ala residue and (3) modifications of the Trp residue obtained by replacing the nitrogen atom in the indol ring by either an oxygen ([Bfa⁵]CCK-8) or a sulphur atom ([Bta⁵]CCK-8). Among these different CCK derivatives, [βAla⁷], [ Met⁶] and [ Trp⁵]CCK-8 were not hydrolyzed by enkephalinase: [ Ala^d]CCK-8 was rapidly cleaved by the enzyme. [Bta⁵] and [Bfa⁵]CCK-8 did not prove to be quite resistant; however the C-terminal tetrapeptides having the same modifications on the Trp residue were not cleaved although they interacted with the enzyme binding site. The stability and biological activity of the peptidase-resistant analogues of CCK-8 remain to be determined in vivo.     

八肽胆囊收缩素减轻肿瘤坏死因子诱导的离体兔肺动脉反应性变化及内皮细胞损伤

为探讨八肽胆囊收缩素（ＣＣＫ－８）缓解内毒素休克时肺动脉高压的作用机制,应用离体血管环张力测定技术及扫描电镜方法,观察了ＣＣＫ－８对肿瘤坏死因子α（ＴＮＦ－α）引起的肺动脉反应性及肺动脉内皮细胞超微结构变化的影响。结果显示：离体脑动脉经ＴＮＦ－α（４０００Ｕ／ｍｌ）孵育２ｈ对苯肾上腺素（ＰＥ）的收缩反应、对乙酰胆碱（ＡＣｈ）及硝普钠（ＳＮＰ）的内皮依赖性及非内皮依赖性舒张反应均无明显影响。ＴＮＦ－     

Synaptosomal Membrane-Bound Form of Endopeptidase-24.15 Generates Leu-Enkephalin from Dynorphin 1-8, α- and β-Neoendorphin, and Met-Enkephalin from Met-Enkephalin-Arg6-Gly7-Leu

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.     

Cholecystokinin octapeptide analogues stable to brain proteolysis

Based on recent findings identifying the initial degradative cleavage of CCK-8 at the Met3-Gly4 bond by a metalloendopeptidase, two analogues of CCK-8 with D-Ala and D-Trp substitutions at the Gly4 position were synthesized as stable analogues. Their stability to proteolysis by brain membranes and their binding potency at central CCK receptors were quantified. Both peptides are stable to degradation by peptidases in cortical synaptic membrane preparations. The analogues are nearly equipotent to CCK-8 in their affinities for inhibition of 125I-CCK-33 binding to guinea pig cortical membranes. L-Ala and L-Trp substituted peptides were synthesized for comparison. Both these peptides are degraded by synaptic membranes and the L-Trp substituted peptide possesses a greatly reduced affinity for central CCK receptors. Therefore, the structure of CCK due to the D conformation of Gly is more capable of interacting with brain CCK receptors. Further conformational analysis will establish whether the stabilized structure is a beta-bend or a beta-turn. Since these peptides are highly potent and stable to brain proteolysis they may be useful as stable CCK analogues for in vivo application.     

The role of calcium in agonist-stimulated hydrolysis of phosphatidylinositol in mouse pancreas

The effects of Ca²⁺ on agonist-stimulated hydrolysis of myo-[2-³H]inostol-labelled phosphatidylinositol in mouse pancreas in vitro, were studied. The increase in cytosol Ca²⁺ concentration produced by the ionophore A23187 did not stimulate the breakdown of phosphatidylinositol. Cholecystokinin-octapeptide (CCK-8) stimulated the hydrolysis of phosphatidylinositol under conditions in which intracellular calcium stores were depleted. The breakdown of phosphatidylinositol was stimulated by bethanechol and CCK-8 in Ca²⁺-free Krebs solution, and the addition of Ca²⁺ to the medium potentiated the effects of these agonists. Lanthanum significantly reduced bethanechol and CCK-8 stimulated hydrolysis of phosphatidylinositol in Krebs solution, but was without effect in Ca²⁺-free Krebs solution. The results of this study support the proposal that PI hydrolysis does not occur as a result of Ca²⁺ mobilization and may be involved in Ca²⁺ gating in the pancreas.