Effect of somatostatin on postprandial gallbladder relaxation.

Although the inhibitory effect of somatostatin (SST) on gallbladder contraction is well known, the influence of SST on gallbladder motility during the late postprandial or relaxation phase has not been studied. We therefore investigated the effect of SST on gallbladder relaxation and gut hormone release during the late postprandial phase. Eight healthy volunteers participated in two experiments performed in random order during continuous infusion of either SST or saline (placebo) starting 2 h after meal ingestion. At regular intervals, gallbladder volumes were measured (ultrasonography) and blood samples were taken for determination of plasma cholecystokinin (CCK), pancreatic polypeptide (PP), peptide YY (PYY) and neurotensin levels (radioimmunoassay). Postprandial gallbladder contraction was similar in both experiments: 68 +/- 4% vs. 66 +/- 4%. During SST infusion, postprandial gallbladder contraction was significantly (P<0.01) reduced (2874 +/- 813% *240 min) compared with saline (9391 +/- 1595% *240 min). Plasma CCK, PP, PYY and neurotensin levels were in the same range in the early postprandial phase but were significantly reduced during SST infusion compared with placebo (late postprandial phase). Plasma levels of CCK correlated with gallbladder volumes during both the contraction and relaxation phase (r=0.68, P=0.01 and r=0.61, P=0.008, respectively). SST enhances gallbladder relaxation and reduces hormone secretion in the late postprandial phase. The results point to an association between CCK and gallbladder volume not only during the postprandial contraction phase but also during the relaxation phase.     

CCK mediated the inhibitory effect of oxytocin on the contraction of longitudinal muscle strips of duodenum in male rats

The aim of the present study was to investigate the effect of oxytocin (OT) on duodenum motility in rats and the possibility that cholecystokinin (CCK) was involved in this process. The isometric contraction of longitudinal muscle strips of duodenum was monitored by polygraph. ELISA was used to measure the concentration of CCK and OT in duodenum. CCK mRNA was assayed by RT-PCR. Oxytocin receptor (OTR) and CCK in duodenum were located by immunohistochemistry and immunofluorescence staining. OT (10^?5 and 10^?6 M) inhibited the spontaneous contraction of the muscle strips. On the contrary, atosiban (OT receptor antagonist), lorglumide (CCK₁ receptor antagonist), and tetrodotoxin (TTX, blocker of voltage-dependent Na⁺ channel on nerve fiber) excited the contraction. The inhibitory effect of OT on duodenal motility was reversed by pretreatment of atosiban, lorglumide, or TTX. Exogenous OT did not influence the expression of OT mRNA in duodenum but increased the concentration of CCK in the culture medium of the cells isolated from longitudinal muscle myenteric plexus. The OTR and CCK were co-expressed in the neurons of the myenteric plexus in duodenum. We concluded that OT inhibited the contraction of the LD spontaneous contraction of rats in vitro. This effect was mediated by the CCK released from the neurons of the myenteric plexus in duodenum.     

Hyperoxia reduces basal release of nitric oxide and contracts porcine coronary arteries

Aim: The purpose of the present study was to investigate whether changes in nitric oxide (NO) concentration is involved in hyperoxia‐induced vasoconstriction in porcine conduit coronary arteries. Methods: The effect of hyperoxia on NO release and vasoconstriction was evaluated by tension recording, microsensor measurements, and immunoblotting in porcine conduit coronary arteries contracted with U46619 or 5‐hydroxytryptamine. Results: In endothelium‐intact segments exchanging 20% O₂, 5% CO₂, 75% N₂ (normoxia) for 95% O₂, 5% CO₂ (hyperoxia) increased contraction. In segments without endothelium hyperoxia‐evoked contraction was abolished, but restored by an encircling donor segment with endothelium. An inhibitor of NOS, asymmetric dimethylarginine (ADMA, 300 μm ), reduced hyperoxic contraction and basal NO concentration by, respectively, 38 ± 12% and 46 ± 3% (P < 0.05, n = 9). A NO donor, S‐nitroso‐N‐acetylpenicillamine (SNAP), increased NO concentration and evoked relaxation to the same levels in normoxic and hyperoxic conditions. β‐actin and endothelial NO synthase (eNOS) protein expression was similar in normoxic and hyperoxic arterial segments. Phosphorylation of eNOS was unaltered in normoxia vs. hyperoxia, but phosphorylation of eNOS‐Ser¹¹⁷⁷ was increased and phosphorylation of eNOS‐Thr⁴⁹⁵ decreased by U46619. Blockers of ATP‐sensitive, voltage‐dependent and calcium‐activated K⁺ channels did not change hyperoxic contraction. However, high extracellular K⁺ concentration or a second and third exposure to hyperoxia decreased contraction. Conclusion: The present study provides direct evidence that hyperoxia reduces basal release of NO leading to depletable endothelium‐dependent vasoconstriction in porcine coronary arteries independent of changes in eNOS phosphorylation.     

A Mechanical Model for CCK-Induced Acalculous Gallbladder Pain

This study investigates the potential correlation between acalculous biliary pain and mechanical stress during the bile-emptying phase. This study is built on the previously developed mathematical model used to estimate stress in the gallbladder wall during emptying [Li, W. G., X. Y. Luo, et al. Comput. Math. Methods Med. 9(1):27-45, 2008]. Although the total stress was correctly predicted using the previous model, the contribution from patient-specific active stress induced by the cholecystokinin (CCK) test was overlooked. In this article, we evaluate both the active and passive components of pressure in a gallbladder, which undergoes isotonic refilling, isometric contraction and emptying during the infusion of CCK. The pressure is estimated from in vivo ultrasonographical scan measurements of gallbladder emptying during CCK tests, assuming that the gallbladder is a thin ellipsoidal membrane. The passive stress is caused by the volume and shape changes during refilling at the gallbladder basal pressure, whereas the active stress arises from the pressure rise during the isometric gallbladder contraction after the CCK infusion. The effect on the stress estimates of the gallbladder to the liver is evaluated to be small by comparing numerical simulations of a gallbladder model with and without a rigid 'flat top' boundary. The model was applied to 51 subjects, and the peak total stress was found to have a strong correlation with the pain stimulated by CCK, as measured by the patient pain score questionnaires. Consistent with our previous study for a smaller sample, it is found that the success rate in predicting of CCK-induced pain is over 75%.     

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

 Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840–G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 μM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 μM) and cholecystokinin-(26-33)-peptide amide (CCK₈, 0.1 μM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK₈-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-α and, to a lesser extent, PKC-ɛ. In addition, TPA-induced inhibition of the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-α. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca²⁺]_i. Taken together, the present findings are in agreement with the idea that PKC-α-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca²⁺]_i oscillations and eventually to inhibition of high-affinity Ca²⁺ signalling through the native CCK receptor in pancreatic acinar cells. Received: 15 September 1997 / Accepted: 2 October 1997     

Interaction of gastrin I, secretin, and cholecystokinin on gallbladder smooth muscle.

The effect of cholecystokinin (CCK), the octapeptide of cholecystokinin (CCK-OP), gastrin I, and secretin was studied on guinea pig gallbladder smooth muscle in vitro. Both CCK and CCK-OP stimulated gallbladder contraction, with CCK-OP being more potent. Gastrin I, over a wide dose range, had no effect on gallbladder contractility. Secretin alone also showed no effect on gallbladder smooth muscle but in combination with CCK-OP it produced a noncompetitive type of inhibition. Michaelis-Menten kinetics showed the calculated maximum response of the secretin plus CCK-OP interaction to be less than with CCK-OP alone. There was no change in the dose required to achieve half-maximal response, D50. These studies indicate that: a) CCK-OP has a greater effect on gallbladder contractility than CCK, b) gastrin I has no effect on gallbladder muscle tone, and c) secretin acts as a noncompetitive antagonist of CCK-OP. These findings suggest that gallbladder motor function may be determined in part by the interaction of secretin and CCK rather than solely in response to CCK.     

Role of spinal cholecystokinin in neuropathic pain after spinal cord hemisection in rats

In the present study we determined whether spinal cholecystokinin (CCK) or the cholecystokinin receptor is involved in below-level neuropathic pain of spinal cord injury (SCI). The effect of the CCK_B receptor antagonist, CI-988 on mechanical allodynia and the expression level of CCK and CCK_B receptor were investigated. Spinal hemisection was done at the T13 level in rats under enflurane anesthesia. CI-988 was administered intraperitoneally and intrathecally and behavioral tests were conducted. After systemic injection, mechanical allodynia was reduced by higher doses of CI-988 (10 and 20 mg/kg). Intrathecal CI-988 (100, 200 and 500 μg) dose-dependently increased the paw withdrawal threshold in both paws. Following spinal hemisection, CCK mRNA expression increased on the ipsilateral side at the spinal segments caudal to the injury and both sides of the spinal L4-5 segments without any significant changes in CCK_B receptor mRNA levels. These results suggest that up-regulation of spinal CCK may contribute to maintenance of mechanical allodynia following SCI and that clinical application of CI-988 or similar drugs may be useful therapeutic agents for management of central neuropathic pain.     

Intramuscular pressure and tissue oxygenation during low‐force static contraction do not underlie muscle fatigue

Aim: To test the hypothesis that time‐wise increase in intramuscular pressure (IMP) and subsequent decrease in muscle tissue oxygenation (TO₂) results in muscle fatigue development during a non‐exhaustive, low‐force contraction evidenced by changes in electromyogram (EMG) and particular mechanomyogram (MMG). Methods: Seven subjects performed static elbow flexion at 10% maximal voluntary contraction (MVC) for 10 min (10% MVC_{10 min}). Surface EMG, MMG, IMP and TO₂ measured by near‐infrared spectroscopy was recorded from m. biceps brachii during 10% MVC_{10 min} and during 5% MVC test contractions of 1 min duration performed before 10% MVC_{10 min}, 10 and 30 min post‐exercise. EMG and MMG were analysed for root mean square (rms) and mean power frequency (mpf). Results: During 10% MVC_{10 min} MMGrms increased from initial level of 0.04 ± 0.01 to 0.11 ± 0.07 m s⁻² in the last minute and MMGmpf and EMGmpf decreased from 34.9 ± 8.2 to 21.3 ± 3.8 Hz and from 71.7 ± 10.9 to 61.7 ± 10.0 Hz respectively. Similar changes were present in 5% MVC test contractions 30 min post‐exercise. Initially, TO₂ decreased by 6.9 ± 6.5% of resting level but returned to rest within 1 min. IMP remained constant during the contraction after an initial fourfold increase from resting level of 12.2 ± 10.4 mmHg. Conclusions: IMP was anticipated to increase with time of contraction due to e.g. increased muscle water content; but this was not confirmed. Consequently, muscle blood flow was unlikely to be impeded with contraction time, which may account for the maintenance of TO₂. Thus, decreased TO₂ did not underlie either acute or long‐term muscle fatigue development evidenced by changes in EMG and particular MMG variables.     

凝集反应对大鼠组织因子、D-二聚体及内皮细胞的影响

目的:探讨血凝素诱发的凝集反应对血浆组织因子(TF)、D-二聚体及内皮细胞的影响。方法:42只SPF级SD大鼠(雌雄不限,体重180~200 g),随机分为生理盐水对照组、不同浓度(5、10和20 g/L)植物血凝素组及灭活的植物血凝素组,每组6只。通过尾静脉向大鼠体内注入生理盐水、植物血凝素及灭活植物血凝素,用ELISA法检测大鼠血浆TF和D-二聚体的水平,透射电镜观察大鼠肺部毛细血管内皮细胞形态。结果:5、10和20g/L植物血凝素组大鼠血浆TF和D-二聚体含量均显著高于生理盐水对照组及相应浓度的灭活植物血凝素组(P0.05)。电镜下,与生理盐水对照组相比,各浓度植物血凝素组出现肺毛细血管内皮细胞损伤,表现为内皮细胞肿胀、溶解,胞质疏松,边界不清,线粒体肿胀,基膜增厚。各灭活植物血凝素组内皮细胞结构正常。结论:体内发生的凝集反应可破坏毛细血管内皮细胞,导致凝血纤溶系统紊乱。     

Receptor-evoked Ca2+ mobilization in pancreatic acinar cells: Evidence for a regulatory role of protein kinase C by a mechanism involving the transition of high-affinity receptors to a low-affinity state

In order to establish a regulatory role for phosphoproteins in the process of receptor-stimulated Ca²⁺ mobilization, isolated pancreatic acinar cells, loaded with fura-2, were stimulated with cholecystokin-in-octapeptide (CCK₈) in the presence of either staurosporine, a general inhibitor of protein kinase activity, or 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C. Staurosporine alone did not affect the average free cytosolic Ca²⁺ concentration ([Ca²⁺]_i,av) in a suspension of acinar cells. However, in the presence of 1.0 M Staurosporine the stimulatory effect of submaximal concentrations of CCK₈ was significantly enhanced. The potentiating effect of the inhibitor was paralleled by the increased production of inositol 1,4,5-trisphosphate. In addition, staurosporine evoked a transient increase in [Ca²⁺]_i,av in cells prestimulated with a submaximal concentration of CCK₈. The data obtained with staurosporine indicate that CCK₈-stimulated phosphorylations exert a negative feedback role in the process of receptor-mediated Ca²⁺ mobilization. The involvement of protein kinase C was investigated by studying the effects of TPA on CCK₈-induced Ca²⁺ mobilization. The phorbol ester induced a rightward shift of the dose/response curve for the CCK₈-evoked increase in [Ca²⁺]_i,av, which, in contrast to the unlimited shift obtained with the receptor antagonist D-lorglumide, reached a maximum of approximately one order of a magnitude at 10 nM TPA. The inhibitory effect of TPA was completely overcome by CCK₈ at concentrations at or beyond 10 nM. This observation has led to the hypothesis that protein kinase C, directly or indirectly, converts the CCK receptor from a high-affinity state to a low-affinity state. Substantial evidence in favour of this hypothesis was provided by the observation that the increase in [Ca²⁺]_i,av evoked by the CCK₈ analogue JMV-180, which acts as an agonist at the high-affinity receptor, was completely blocked by TPA pretreatment. TPA also evoked a rightward shift of the dose/response curve for the carbachol-induced increase in [Ca²⁺]_i,av, indicating that the protein-kinase-C-mediated transition of the affinity state of receptors is a more general phenomenon. In the presence of submaximal CCK₈ concentrations, TPA dose-dependently decreased the poststimulatory elevated [Ca²⁺]_i,av to the prestimulatory level, indicating that protein kinase C also inhibits the process of sustained Ca²⁺ mobilization. The effects of TPA were counteracted by staurosporine, suggesting that the effects of the inhibitor itself were indeed due to inhibition of the receptor-mediated activation of protein kinase C. The data presented are in support of a negative-feedback role for protein kinase C in the process of receptor-mediated Ca²⁺ mobilization by a process that involves phosphorylation of the CCK receptor, thereby transforming it from a high-affinity state into a low-affinity state.     

Plasma cholecystokinin levels in Prader‐Willi syndrome and obese subjects

The cardinal feature of individuals with Prader‐Willi syndrome (PWS) is severe hyperphagia‐mediated obesity resulting from a faulty satiety mechanism. PWS is the most common genetic cause of marked obesity. Cholecystokinin (CCK) is a 33‐amino‐acid peptide found in high levels in the gut and brain involved in mediating the satiety response to meals. Free fatty acids (FFA) are responsible for the stimulation of CCK release after a fatty meal, and CCK and plasma FFA levels rise in tandem in normal individuals. Fasting plasma CCK levels were measured by radio‐immunoassay in 33 PWS subjects with a mean age of 22.2 years ± 8.1 years and 24 obese control subjects without a known cause of their obesity with a mean age of 28.7 years ± 12.9 years. Consistent with previous findings, neither fasting plasma FFA levels (617.5 versus 486.8 μm/mL) or CCK levels (21.0 versus 19.1 pg/mL) were significantly different in PWS or control subjects, respectively. However, there was a significant correlation between fasting plasma FFA and CCK levels in obese subjects (r = 0.64, P < 0.01), this correlation was completely lacking in PWS subjects (r = −0.06, P = 0.79). This difference in correlation coefficients constitutes a large effect. There were no significant effects observed for genetic subtypes (15q11‐q13 deletion or maternal disomy 15), body mass index, percentage of fat, plasma levels of insulin, C‐peptide, glucagon or leptin, age, or gender on CCK levels in our PWS subjects. These results suggest that differences in the peripheral CCK response to FFA levels may be a factor contributing to the altered satiety response in PWS subjects. Am. J. Med. Genet. 95:67–70, 2000. © 2000 Wiley‐Liss, Inc.     

Effects of cholecystokinin on Y, X, and W cells in the dorsal lateral geniculate nucleus of rats

The role of the cholecystokinergic input to the rat's dorsal lateral geniculate nucleus (dLGN) was studied by examining the effect of iontophoretically administered CCK-8S on the neuronal response to stimulation of the receptive field center. Peristimulus activity was recorded extracellularly from 108 neurons grouped according to the type of receptive field (OFF, ON, or ON-OFF) and classified with respect to their Y, X, or W properties by means of discriminant analysis. CCK affected the response to a center-sized spot of light in two thirds of the neurons investigated. The center response decreased in 50 of 73 CCK-sensitive neurons (69%), predominantly in Y OFF and X OFF center cells (17 of 19). In the remaining 23 cells the center response increased, most consistently (11 of 17) in W ON center cells. Center and surround responses were similarly influenced. Inhibitions and excitations induced by CCK-8S were reproducible, dose dependent, and receptor mediated. The CCK_B antagonist PD 135158 reduced the CCK effects in 10 of 14 cells; the CCK_A antagonist KL 1001 reduced the CCK effects in 17 of 36 cells. The CCK-induced inhibition was B-receptor specific in 4 of 8 cells, A-receptor specific in 2 of 8 cells, and partially mediated by each of the two types of receptor in the remaining 2 cells. Blocking by the CCK_A antagonist was more frequently observed in W cells than in cells with Y or X characteristics. The data show that CCK modifies the activity of dLGN cells in a variable direction depending on the specific cell type (Y, X, W) and response pattern (OFF, ON). The effects of CCK are discussed in relation to proposed functions of the superior collicular input to the dLGN.     

Muscle performance following fatigue induced by isotonic and quasi‐isometric contractions of rat extensor digitorum longus and soleus muscles in vitro

Aim: To study the effect of contraction mode on fatigue development. Methods: Muscle fatigue was induced by isotonic and quasi‐isometric contractions in rat soleus (SOL) and extensor digitorum longus (EDL) muscles, using identical stimulation protocol (60 Hz, 400 ms s^?1) for 100 s in SOL and 60 s in EDL. Fatigue was quantified as the decline in peak values of shortening, shortening velocity, relaxation and work during the isotonic contractions, and, correspondingly, of force, rate of force development, relaxation and work during the quasi‐isometric contractions. Maximal test contractions (60 Hz, 1.5 s) performed before and after fatigue were analysed for decline in force development (F_max), rate of force development (dF/dt_max) and relaxation (?dF/dt_max). Results: F _max declined to significantly lower values after isotonic than after quasi‐isometric fatiguing contractions (fatigued in percentage of unfatigued): 58.5 ± 6.4% vs. 64.4 ± 7.0% in SOL, and 30.4 ± 4.1% vs. 33.3 ± 3.6% in EDL, respectively. The same pattern was seen for dF/dt_max which decreased to: 46.3 ± 9.9% vs. 52.3 ± 8.5% in SOL, and 19.1 ± 4.3% vs. 22.3 ± 3.2% in EDL after isotonic and quasi‐isometric contractions, respectively. Similarly, when comparing fatigue development during the two contraction modes, the respective fatigue variables decreased more rapidly and to lower levels during isotonic vs. quasi‐isometric contractions. During maximal test contractions, the dynamic fatigue variables (±dF/dt_max) declined to significantly lower levels than F_max. Conclusions: Fatigue development was significantly larger during isotonic vs. quasi‐isometric contractions. The use of force as the only experimental fatigue variable may underestimate the functional impairment of fatigued muscle, neglecting the fatigue effect on time and length dimensions.     

CCKB受体诱发的小鼠神经元蛋白质磷酸化的研究

目的：从蛋白质可逆磷酸化修饰的角度初步勾勒出CCK_B型受体介导的胞内信号转导途径。方法:培养的小鼠大脑皮质神经元分为实验组、对照组和受体拮抗剂组，在无磷培养基加入 ［32P］-NaH₂PO₄标记细胞中的磷蛋白后，刺激组给予CCK₈（10^-7 mol/L），对照组加入等量的无磷培养基，受体拮抗剂组在分别加入CCK_A型、CCK_B型受体拮抗剂L364 718、L365 260以及L364 718＋L365 260，浓度均为10^-8 mol/L，孵育10 min后，再加入10^-7 mol/L CCK₈，37 ℃作用60 min，液氮中终止磷酸化反应。裂解细胞提取蛋白质，双向电泳分离，放射自显影7d后，获得磷酸化蛋白的放射自显影双向电泳图谱。采用PDQuest 2D分析软件对图谱进行差异分析，并在Swiss-Prot蛋白质数据库和自建磷蛋白数据库中查询定性。结果:CCK₈作用60 min后，小鼠神经元CCK₈信号转导相关磷酸化蛋白有：多种蛋白激酶、细胞信号分子、生长因子受体、转录因子等。加入L364 718后，神经元中PKCδ、P55G等的磷酸化水平降低，加入L365 260后， PKCα、PKGβ、OGFR、EGFR等的磷酸化水平呈现不同程度的降低，表明皮质神经元中CCK_B受体介导的信号转导更复杂。结论:CCK_A型、GGK_B型受体均可介导CCK₈在神经元的信号转导，但CCK_B型受体可能发挥了更重要的作用，其介导的信号途径可能包括：肌醇磷脂信使系统、cAMP-PKA途径、MAPK途径、JNK途径、PI3K-PKB途径和cGMP-PKG途径。     

Prolonged asthmatic responses to inhaled methacholine

The pattern of asthmatic response after inhalation of atropine and methacholine was studied in six adult asthmatics. After pretreatment with atropine, the provocation concentration of methacholine to cause a fall in FEV₁ of 20% was increased from 0.66 ± 2.09 to 94.90 ± 1.78 mg/ml. In the subsequent 7 hr, four subjects developed prolonged asthmatic responses. These occurred after concentrations of methacholine higher than those used clinically but did not directly relate to the dose of methacholine or to the increase in dose after atropine. In one subject the prolonged response was not accompanied by increased methacholine responsiveness and was not prevented by pretreatment with cromolyn sodium (40 mg). These results show that high doses of methacholine inhaled after pretreatment with atropine can induce prolonged asthmatic responses but the mechanism is unclear.     

Cholecystokinin-stimulated enzyme secretion from dispersed rabbit pancreatic acinar cells: phosphorylation-dependent changes in potency and efficacy

In order to establish a regulatory role for phosphoproteins in receptor-stimulated enzyme secretion, dispersed rabbit pancreatic acinar cells were stimulated with the COOH-terminal octapeptide of cholecystokinin (CCK₈) in the absence and presence of staurosporine and/or 12-O-tetradecanoylphorbol 13-acetate (TPA) or forskolin. The dose/response curve for the stimulatory effect of CCK₈ on amylase secretion was biphasic, with a mean half-maximal concentration (EC₅₀) of 21 pM. Staurosporine (1 M) did not affect secretion elicited by CCK₈ concentrations below 0.1 nM, but reduced the response to CCK₈ concentrations above 0.1 nM. As a result, the mean EC₅₀ for CCK₈ decreased to 8 pM and its efficacy to 70%. The phorbol ester TPA (0.1 M) attenuated secretion evoked by CCK₈ concentrations below 0.1 nM and potentiated the response to CCK₈ concentrations above 0.1 nM. As a result, the mean EC₅₀for CCK₈ increased to 0.14nM and its efficacy to 300%. Staurosporine abolished both the inhibitory and the potentiating effect of TPA, thereby turning the inhibitory effect into a strong potentiating effect. As a result, the mean EC₅₀ for CCK₈ decreased to 3 pM, whereas its efficacy increased to 190%. Forskolin (30 M) potentiated the response to both the lower and the higher CCK₈ concentrations. As a result, the mean EC₅₀ for CCK₈ increased to 28 pM and its efficacy to 300%. Staurosporine enhanced the potentiating effect of forskolin at CCK₈ concentrations below 0.1 nM, but abolished potentiation at CCK₈ concentrations above 0.1 nM. As a result, the mean EC₅₀for CCK₈ decreased to 1.4 pM, whereas its efficacy increased to 260%. The data presented demonstrate that the apparent sensitivity of dispersed pancreatic acinar cells to stimulation of the process of enzyme secretion by CCK₈ decreases when kinases are activated and increases when kinases are inactivated. Moreover, they show that the efficacy of CCK₈ increases by the action of kinases, both sensitive and insensitive to staurosporine.     

The Effect of Jeo Dang‐Tang on Cytokines Production in the Patients with Cerebral Infarction

Abstract

The herbal formulation “Jeo Dang‐Tang” (JDT) has long been used for various cerebrovascular diseases. However, very little has scientific investigation been carried out. The aim of the present study is to investigate the effect of JDT on the production of various cytokines in the patients with cerebral infarction (CI). Peripheral blood mononuclear cells (PBMC) obtained from the patients with CI were cultured for 24 h in the presence or absence of lipopolysaccharide (LPS) or phytohemagglutinin (PHA). The amount of interleukin (IL)‐4, IL‐10 and transforming growth factor (TGF)‐1β, in culture supernatant, was significantly increased in the JDT, LPS or PHA treated cells compared to unstimulated cells (P < 0.05). We also show that increased IL‐4, and IL‐10 level by LPS or PHA was significantly inhibited by JDT in a dose‐dependent manner. Maximal inhibition rate of IL‐4 and IL‐10 production by JDT was 45 ± 2% and 51 ± 5% for LPS‐stimulated cell and 41.5 ± 3% and 70.8 ± 2% for PHA‐stimulated cells, respectively (P < 0.05). On the other hand, JDT significantly increased the LPS or PHA‐induced TGF‐β1 production (P < 0.05). These data suggest that JDT has a regulatory effect on the cytokines production, which might explain its beneficial effect in the treatment of CI.     

Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failure

Introduction: Vasopressin (AVP) stimulates sodium reabsorption and Na,K,2Cl‐cotransporter (NKCC2) protein level in the thick ascending limb (TAL) of Henle's loop in rats. Rats with congestive heart failure (CHF) have increased protein level of NKCC2, which can be normalized by angiotensin II receptor type‐1 (AT₁) blockade with losartan. Aim: In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT₁ receptor blockade on this system. Method: CHF was induced by ligation of the left anterior descending coronary artery (LAD). SHAM‐operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day^?1 i.p.). Results: CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10^?6 m ) stimulated cAMP levels were significantly increased in CHF rats (25.52 ± 4.49 pmol cAMP μg^?1 protein, P < 0.05) compared to Sham rats (8.13 ± 1.14 pmol cAMP μg^?1 protein), P < 0.05). Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 ± 1.93 fmol μg^?1 protein vs. Los‐CHF: 7.49 ± 1.08, P < 0.05), but not in Sham rats (SHAM: 4.66 ± 0.59 vs. Los‐SHAM: 4.75 ± 0.71). AVP‐mediated cAMP accumulation was absent in both groups treated with losartan (Los‐SHAM: 4.75 ± 0.71 and Los‐CHF: 7.49 ± 1.08). Conclusion: The results indicate that the increased NKCC2 protein level in the mTAL from CHF rats is associated with increased cAMP accumulation in this segment. Furthermore, the finding that AT₁ receptor blockade prevents AVP‐mediated cAMP accumulation in both SHAM and CHF rats suggests an interaction between angiotensin II and AVP in regulation of mTAL Na reabsorption.     

Development of an immune function assay by measuring intracellular adenosine triphosphate (iATP) levels in mitogen-stimulated CD4+ T lymphocytes

We developed an immune function assay for monitoring CD4+ T cells activity based on changes in intracellular adenosine triphosphate (iATP) levels after phytohemagglutinin (PHA) stimulation. Blood samples were obtained from 40 healthy subjects and 30 RTRs and incubated with 5 µg/mL of PHA for 15–18 hr at 37°C and 5% CO₂. Afterward, the CD4+ T cells were separated by antibody-coated magnetic beads and lysed. Then, iATP content in unstimulated and stimulated conditions was measured by luciferin-luciferase reaction using a log-log standard curve. The iATP levels showed significant increase in CD4+ T cells in both healthy persons (mean: 550 ± 142 ng/mL vs. 109 ± 54 ng/mL) and RTRs (mean: 394 ± 160 ng/mL vs. 52 ± 37 ng/mL) after PHA stimulation (P < 0.001). However, the iATP production in RTRs was significantly lower than that in healthy individuals; both prior to and after stimulation with PHA (P < 0.001). No gender-specific difference in iATP production was observed between women and men subjects. This rapid and low-cost assay reflects the degree of immune cell function through assessment of CD4+ T cells activation. Thus, it can be used for evaluation of immune system status in immunodeficient individuals as well as in immunosuppressed transplant recipients who needs drug adjustment.     

Renal and hormonal responses to isotonic saline infusion after 3 days' head‐down tilt vs. supine and seated positions

Aim: The study aimed to determine whether prolonged exposure to simulated microgravity produces a level of thoracic volume receptor loading similar to that seen in the upright position or immediately after lying down. Methods: We used a cross‐over design to compare responses to a saline infusion in eight healthy subjects during a 4‐day, ?6° head‐down tilt (HDT) and in the acute seated and acute supine positions. Results: The first 24 h of HDT were associated with greater urinary excretion of water and sodium (UV, UNaV) than seated and acute supine [cumulative UV, 3035 ± 219, 2311 ± 156 (P < 0.05), and 2448 ± 182 mL (P < 0.05), respectively; cumulative UNaV, 256 ± 19, 180 ± 11 (P < 0.05), and 189 ± 15 mmol (P < 0.05), respectively]. Haemoglobin and haematocrit were increased after 24 h and plasma volume decreased after 48 h of HDT (P < 0.05). With prolongation of HDT, UV and UNaV returned near the baseline values, and plasma atrial natriuretic factor (ANF) and renin values returned to acute seated levels; in acute supine, ANF values were higher and renin lower than in the two other positions. After a 30‐min infusion of 20 mL kg^?1 isotonic saline on the fourth HDT day or during acute seated or acute supine, sodium excretion within 4 h was similar during HDT and acute seated (83 ± 6 and 84 ± 9 mmol, respectively) and greater during supine (104 ± 8 mmol, P < 0.05). The renin decrease was greater in HDT and seated than in supine. The plasma ANF increase was greater during HDT than during supine; during seated, plasma ANF was unchanged. Conclusion: These data suggest that, after 4 days of HDT, thoracic volume receptor loading returns to the same level as in the seated position, leading to blunted responses to volume expansion as compared with the acute supine position.