Evidence for an agonist-induced, ATP-dependent change in muscarinic receptors of intact 1321N1 cells

The binding of muscarinic agonists, partial agonists and antagonists to muscarinic receptors of intact 1321N1 human astrocytoma cells and of cell lysates was studied. Partial agonists and antagonists exhibited similar apparent affinities in intact cell competition binding assays with either the lipophilic radioligand [3H]quinuclidinyl benzilate [( 3H]QNB) or the hydrophilic radioligand [3H]N-methyl scopolamine [( 3H]NMS). In contrast, full agonists exhibited markedly lower apparent affinities in intact cell competition binding assays with [3H]QNB than with [3H]NMS. The affinities of agonists and antagonists in competition for [3H] NMS binding to intact cells were slightly higher than those observed in competitions for [3H]QNB binding in cell lysates. In contrast, the affinities of agonists in competition for [3H]QNB binding to intact cells were considerably lower than those in competition for [3H]QNB binding in cell lysates. Treatment of cells with antimycin A to deplete intracellular ATP prevented agonist-induced internalization of muscarinic receptors as assessed by sucrose density gradient assays of receptor subcellular distribution. In ATP-depleted cells, the apparent affinities of full agonists vs. [3H]QNB were markedly higher and were similar to their apparent affinities vs. [3H]QNB in cell lysates. The apparent affinities of partial agonists and of antagonists were unaffected by ATP depletion. ATP depletion decreased slightly the apparent affinity of the full agonist carbachol in competition for [3H]NMS binding to intact cells, whereas the apparent affinity of atropine was unchanged. These results provide evidence for an agonist-specific, ATP-dependent low affinity state of intact cell muscarinic receptors that may be similar to those observed previously for both beta and alpha-1 adrenergic receptors and that may be related to receptor internalization/sequestration.     

Effects of dichlorobenzamil on calcium currents in clonal GH3 pituitary cells

Membrane currents were recorded from voltage-clamped clonal (GH3) pituitary cells, using the whole-cell patch clamp method. Under conditions in which currents through Na+ and K+ channels were abolished, two distinct Ca++ channel currents were identified. Dichlorobenzamil (DCB), an amiloride analog and potent inhibitor of Na-Ca exchange, inhibited both the T-type Ca++ current (ICa,t) and slowly-inactivating L-type current carried by either Ca++ (ICa,s) or Ba++ (IBa,s). The blockade was dose-dependent (1-25 microM) and ICa,t was more sensitive to inhibition by DCB than the L-type channel currents. Although the inhibition of ICa,t was not accompanied by changes in the time course of inactivation of the T-type channels, the blockade of L-type Ca++ channel currents was time-dependent, increasing throughout the depolarizing test pulse (300 msec). Repetitive stimulation at 1.0 Hz or the application of depolarizing prepulses augmented the blockade of ICa,s and IBa,s by DCB. It is proposed that the blockade is modulated by the functional state of the L-type channel, being enhanced by the channel opening. Currents conveyed by Na+ ions through both types of Ca++ channels in GH3 cells equilibrated with Ca++-free salines were inhibited by DCB concentrations (1-5 microM) comparable to those required for blocking the currents conveyed by Ca++ or Ba++ ions.     

Ghrelin对GH3细胞膜电位的作用

目的观察ghrelin对GH3细胞膜电位的作用。方法用Nystatin打孔全细胞记录跨膜电流,观察ghrelin组和对照组GH3细胞膜电位。结果对照组细胞的膜电位为-(72±4.78)mV,在ghrelin处理后,膜电位减小到(54.8±4.58)mV。结论ghrlein可以快速、可逆、显著性地降低GH3细胞膜电位。     

Phorbol ester-induced inhibition of cyclic GMP formation mediated by muscarinic receptors in murine neuroblastoma cells

The effects of phorbol 12-myristate 13-acetate (PMA) on carbamylcholine (CBC)-induced [3H]cyclic GMP formation in mouse neuroblastoma cells (clone N1E-115) were studied. PMA, but not 4 alpha-phorbol, suppressed muscarinic receptor-mediated cyclic GMP responses in a time-dependent and a concentration-dependent fashion with an IC50 of 68.8 +/- 20.2 nM. The inhibitory effects of PMA on CBC-induced cyclic GMP formation were of a mixed competitive and noncompetitive type, being characterized by a depression of maximal cyclic GMP response to CBC and a significant increase in its EC50. PMA also significantly reduced [3H]cyclic GMP formation induced by histamine, without affecting the responses elicited either by sodium azide or the calcium ionophore A23187. Although the inhibitory effects of PMA on CBC-induced cyclic GMP formation were not reversed by washing, these effects were significantly attenuated by H-7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], a protein kinase C inhibitor. PMA had no effect on binding of an antagonist ligand to muscarinic receptors, or on the binding characteristics of CBC to these receptors in intact cells. On the other hand, PMA competed for the specific binding of a labeled phorbol ester in intact cells with a potency similar to that of PMA in inhibiting muscarinic receptor-mediated [3H]cyclic GMP responses.     

Effect of contractile activity on muscarinic receptor density and the response to muscarinic agonists

Autonomic receptor density can be modulated by alterations in neuronal activity over a relatively short period of time (hours). The current study investigates whether increased in vivo stimulation of urinary bladder smooth muscle can alter muscarinic receptor density and response to muscarinic stimulation. A high degree of reflex stimulation of the urinary bladder (rabbits) was initiated by stricture of the external urethra. Intravesical pressure and intra-abdominal pressure were monitored continuously over a 4-hr time period. At the end of the 4-hr period, the rabbits were sacrificed and isolated strips of bladder body were either mounted in isolated smooth muscle baths for contractile studies or frozen and stored in liquid nitrogen for muscarinic receptor analysis. These studies demonstrated that over 4 hr of urethral stricture there was a significant reduction in muscarinic receptor density from a Bmax of 34 +/- 3.4 fmol/mg of protein in control bladder strips to 22 +/- 2.4 fmol/mg of protein in the experimental group. In association with the decreased muscarinic receptor density, there was a significant and selective decrease in the contractile response to muscarinic stimulation. Similar to the in vivo studies, repetitive field stimulation of in vitro strips resulted in a significant decrease in muscarinic receptor density and a significant and selective decrease in the contractile response to muscarinic stimulation. The results from these studies indicate that muscarinic receptor density, and response to muscarinic stimulation, can be modulated over a relatively short period of time by alterations in the level of neuronal stimulation.     

Propylbenzilylcholine mustard has greater specificity for muscarinic m2 receptors than for m3 receptors present in cerebellar granule cell culture from rat.

Both muscarinic m2 receptors that inhibit adenylyl cyclase and m3 receptors that stimulate the hydrolysis of inositol phospholipids are expressed in cerebellar granule cells. In order to determine whether a reserve population of either of these receptors is present in this cell culture, the irreversible muscarinic receptor antagonist, propylbenzilylcholine mustard (PBCM), was used at different concentrations to bind various proportions of available muscarinic receptors. After pretreating the cell cultures with low concentrations of PBCM (< 1 nM), the muscarinic m2 receptor-mediated response decreased. Higher concentrations of PBCM (1-3 nM) were needed to reduce the muscarinic m3 receptor-mediated response. These results suggested that either a reserve population of muscarinic m3 receptors is present or that PBCM shows greater specificity for muscarinic m2 receptors. Because the muscarinic m2 receptor is a 66 kDa protein, whereas the muscarinic m3 receptor is a 92 kDa protein, these receptors can be separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis after being labeled with [3H]PBCM. PBCM appears to specifically bind the 66 kDa m2 receptor at concentrations lower than those required to bind to the 92 kDa m3 receptor. A linear correlation was found between the increased binding of [3H]PBCM to each receptor and the proportional loss of that receptor-mediated response. Thus, a reserve population of either muscarinic m2 or m3 receptors does not appear to exist in cerebellar granule cells. These studies also show that PBCM has greater affinity for the muscarinic m2 receptor than the muscarinic m3 receptor.     

Differential control of band 3 lateral and rotational mobility in intact red cells.

Measurements of integral membrane protein lateral mobility and rotational mobility have been separately used to investigate dynamic protein--protein and protein-lipid interactions that underlie plasma membrane structure and function. In model bilayer membranes, the mobilities of reconstituted proteins depend on the size of the diffusing molecule and the viscosity of the lipid bilayer. There are no direct tests, however, of the relationship between mechanisms that control protein lateral mobility and rotational mobility in intact biological membranes. We have measured the lateral and rotational mobility of band 3 in spectrin-deficient red blood cells from patients with hereditary spherocytosis and hereditary pyropoikilocytosis. Our data suggest that band 3 lateral mobility is regulated by the spectrin content of the red cell membrane. In contrast, band 3 rotational mobility is unaffected by changes in spectrin content. Band 3 lateral mobility and rotational mobility must therefore be controlled by different molecular mechanisms.     

Characterization of riluzole-induced stimulation of large-conductance calcium-activated potassium channels in rat pituitary GH3 cells.

BACKGROUND: Riluzole is known to be an inhibitor of glutamatergic neurotransmission. Transmitter release from nerve terminals can be regulated by the activity of large-conductance Ca(2+)-activated K+(BKCa) channels. METHODS: The ionic mechanism of actions of riluzole was investigated in neuroendocrine (GH3 and PC12 cells), using the whole-cell patch-clamp and inside-out excised patch configurations. RESULTS: In GH3 cells, riluzole at 0.3-100 mumol/L increased the amplitude of Ca(2+)-activated K+ current (IK(Ca)) in a concentration-dependent manner with a half maximal concentration of 5 mumol/L. The riluzole-induced increase in outward current was not be suppressed by glibenclamide (10 mumol/L) or apamin (200 nmol/L). However, iberiotoxin (200 nmol/L) or tetrandrine (10 mumol/L) can effectively suppress riluzole-induced IK(Ca). Under inside-out patch recording mode, riluzole (10 mumol/L) applied intracellularly can increase the opening probability of large-conductance Ca(2+)-activated K+(BKCa) channels, but did not affect their single-channel conductance. The riluzole-induced change in the kinetic behavior of BKCa channels is due to an increase in mean open time and a decrease in mean closed time. Riluzole caused a left shift in the midpoint for voltage-dependent opening. Riluzole-stimulated activity of BKCa is independent on internal Ca2+. Riluzole (30 mumol/L) did not affect the amplitude of voltage-dependent K+ current, but it produced a slight reduction of L-type voltage-dependent Ca2+ current. Under current clamp mode, riluzole (10 mumol/L) decreased the firing rate of action potentials induced by thyrotropin releasing hormone (10 mumol/L) in GH3 cells. In rat pheochromocytoma PC12 cells, riluzole also increased the activity of BKCa channels without altering their channel conductance. CONCLUSION: This study shows that riluzole can stimulate the activity of BKCa channel in neuroendocrine cells.     

Effect of amiloride on the intracellular sodium and potassium content of intact Streptococcus faecalis cells in vitro.

Amiloride at millimolar concentrations caused marked changes in the growth-dependent intracellular balance of Na+ and K+ in Streptococcus faecalis. These results, whether specific to transport processes or resulting from indirect yet unknown mechanisms, constitute the first evidence of an effect of amiloride on bacterial electrolytes.     

短时低频电刺激体外培养许旺细胞髓鞘的形成

背景:周围神经系统损伤后,短时低频电刺激已被证明可显著促进轴突再生和选择性功能修复,但目前对电刺激是否影响周围神经髓鞘的形成还知之甚少,而电刺激发挥作用究竟是通过神经元还是许旺细胞还有待证实.目的:建立体外背根神经元与许旺细胞联合培养模型,观察短时低频电刺激对许旺细胞髓鞘形成的影响.方法:体外培养背根神经元,纯化后预先施予电刺激(20 Hz,100 μs,3 V),持续作用1 h,24 h后再加入许旺细胞悬液制成背根神经元/许旺细胞联合培养模型.在此基础上,用L-ascorbic acid诱导髓鞘形成,分别于诱导后第7,14天观察培养体系中髓鞘的形成.结果与结论:电刺激增强背根神经元分泌脑源性神经营养因子(P < 0.05),经电刺激作用的背根神经元再与许旺细胞联合培养,最终表现为髓鞘形成增多以及髓鞘蛋白表达上调(P < 0.05).提示短时低频电刺激对体外许旺细胞髓鞘的形成具有促进作用,初步认为该作用至少通过刺激神经元分泌脑源性神经营养因子增多导致.     

Effect of diazepam on adenosine 3',5'-cyclic monophosphate (cAMP) plasma levels in anesthetized patients

BACKGROUND: It has been previously demonstrated that diazepam inhibits the cyclic nucleotide phosphodiesterase type 4 isozyme (PDE4). PDE enzymes mediate the hydrolysis of the nucleotide adenosine 3',5'-cyclic monophosphate (cAMP). OBJECTIVE: The aim of this study was to determine whether IV administration of diazepam affects cAMP plasma levels in anesthetized patients. METHODS: In this prospective study, patients scheduled to undergo elective myocardial revascularization surgery with anesthetization with etomidate (0.3 mg/kg), fentanyl (total dose 20-25 microg/kg), and cisatracurium (150 microg/kg), supplemented with sevoflurane (2% in an oxygen/air mixture), were randomly assigned to 1 of 3 groups to receive diazepam (0.28 mg/kg IV), diazepam vehicle (alcohol and propylene glycol IV), or saline. Before the start of the surgical procedure, at 5 and 10 minutes after administration of diazepam, vehicle, or saline, blood samples were obtained for determination of the diazepam, cAMP, and catecholamine levels. RESULTS: Ten patients received diazepam, 10 received vehicle, and 5 received saline. The mean (SEM) arterial serum concentrations of diazepam were 2.1 (0.2) microg/mL and 1.1 (0.4) microg/mL, respectively, at 5 and 10 minutes after administration. cAMP plasma levels increased from mean (SEM) baseline values of 30.0 (1.7) nmol/L to 35.5 (1.5) nmol/L (P < 0.05) and 43.1 (1.7) nmol/L (P < 0.05) at 5 and 10 minutes, respectively, after diazepam administration. No significant changes in cAMP plasma levels were observed compared with the mean (SEM) baseline value of 32.0 (1.7) nmol/L at 5 minutes (31.8 [1.3] nmol/L) and 10 minutes (30.9 [1.4] nmol/L) after vehicle administration. Epinephrine plasma concentration increased from a mean (SEM) baseline value of 0.13 (0.02) ng/mL to 0.22 (0.02) ng/mL (P < 0.05) at 10 minutes after administration of vehicle and 0.21 (0.02) ng/mL (P < 0.05) at 10 minutes after administration of diazepam. CONCLUSION: In this preliminary study, diazepam increased cAMP plasma levels in anesthetized patients, presumably through inhibition of PDE4 activity.     

Changes in cAMP formation in mononuclear leukocytes of heart and renal transplant recipients

In mononuclear leukocytes (MNL) of renal transplant recipients treated with cyclosporine A and prednisone, an increase of basal cAMP generation has been observed. In order to characterize the mechanisms underlying changes of cAMP generation in patients who were treated with immunosuppressives following heart transplantation, we investigated the β-adrenoceptor—G protein—adenylate cyclase signal transduction cascade in heart transplant recipients and for comparison in renal transplant recipients as well as controls. Basal cAMP formation in MNL was elevated in heart transplant recipients by 27% and in renal transplant recipients by 148% compared to controls. Following β-adrenoceptor stimulation with isoprenaline, cAMP formation in MNL of heart transplant recipients was similar to the controls, but was enhanced in renal transplant recipients to 138%. Investigation of β-adrenoceptor density on MNL as a possible cause for increased cAMP formation revealed similar receptor numbers in controls and in cardiac or renal transplant recipients. Furthermore, the increase of the β-adrenoceptor density on MNL, which is observed following infusion of isoprenaline, was similar in controls and heart transplant recipients. The amount of pertussis- and cholera toxin substrates was the same in heart transplant recipients as in controls. In contrast, MNL of renal transplant recipients showed a marked increase of G_sα by 45% and a smaller albeit significant increase of G_1α by 15%, as judged by cholera toxin and pertussis toxin labeling, respectively. Investigation of inotropic parameters by echocardiography under control conditions and during the infusion of increasing concentrations of isoprenaline revealed no difference in the basal contractility and the inotropic response to β-adrenergic stimulation in controls and heart transplant recipients. It is concluded that changes of G-protein expression are involved in the increase of the cAMP-generation in MNL of heart transplant recipients. These alterations in MNL cannot be taken as a model of cellular function in the transplanted heart, but it is reasonable to suggest that elevations of cAMP formation in MNL may contribute to the immunosuppressive effects of the treatment with cyclosporine A or corticosteroids, the mechanism of which could be an alteration of G_sα or the catalyst in renal transplant recipients and the catalyst in heart transplant recipients which occurs without any changes of β-adrenoceptors.     

Effects of ethanol in vitro on the beta adrenergic receptor-coupled adenylate cyclase system

The effects of ethanol on the beta adrenergic receptor-coupled adenylate cyclase system were examined in vitro using membranes prepared from S49 lymphoma cells. Ethanol caused a dose-dependent increase in adenylate cyclase activity in membranes prepared from wild-type cells when the activity was measured in the presence of GTP. Activity measured in the presence of isoproterenol was also increased by ethanol, but the fold-stimulation by isoproterenol was lower in the presence of ethanol. Ethanol also shifted the dose-response curve for stimulation of the enzyme by isoproterenol to the right. This shift was due to a decrease in the affinity of the beta adrenergic receptor for isoproterenol. A decrease in the affinity of the receptor for the antagonists [125I]iodopindolol and propranolol was also observed, but the magnitude of this effect was less than that seen with the agonist isoproterenol. The density of binding sites for [125I]iodopindolol was not affected by ethanol. Dose-response curves for NaF and guanosine-5'-O-(3-thiotriphosphate), both of which stimulate adenylate cyclase activity through an effect on the stimulatory guanine nucleotide-binding protein (Gs), were shifted to the left by the addition of ethanol. In membranes prepared from the CYC- variant of S49 cells, which lacks the alpha subunit of Gs, guanosine-5'-O-(3-thiotriphosphate) inhibited forskolin-stimulated adenylate cyclase activity. The inhibition by guanosine-5'-O-(3-thiotriphosphate) was not affected by ethanol. In membranes prepared from both wild-type and CYC- S49 cells, ethanol inhibited forskolin-stimulated adenylate cyclase activity. Whereas the inhibition of this activity by GTP was greatly attenuated in membranes prepared from CYC- S49 cells which had been pretreated with pertussis toxin, the inhibition by ethanol was not affected by pretreatment with pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

腺苷对体外循环下心脏手术患者血浆细胞因子的影响

[目的]探讨腺苷对体外循环心脏手术患者围术期血浆细胞因子的影响.[方法]回顾性分析60例体外循环心脏手术患者的临床资料:A组(33例)为腺苷(cAMP处理组,B组(27例)为等量生理盐水处理组;分别于术前(T0)、主动脉开放10 min(T1)、12 h(T2)、24 h(T3)采集患者动脉血标本,用ELISA法检测围术期血浆TNF-α、IL-8、IL-10、cTnT水平;用自动生化仪测量围术期血浆CK-MB水平.术毕采集右房组织标本,镜下观察肺组织炎症反应.[结果]两组患者术中、后各时点TNF-α、IL-8、IL-10水平均较术前升高(P＜0.05),在T0、T1两组间无明显差异(P＞0.05);在T3、T4两各时点TNF-α、IL-8水平A组较B组明显降低(P＜0.05),而IL-10水平A组较B组明显升高(P＜0.05);术后T2、T3两个时点CK-MB、cTnT水平较术前(T0)明显升高(P＜0.05),但A组较B组明显降低(P＜0.05).镜下观察两组病人均有部分心肌结构破坏,炎症反应,但A组病人较B组轻.[结论]腺苷对缺血再灌注的心肌组织具有一定的保护作用.     

Effect of ultrasonic attenuation on the feasibility of acoustic tweezers

A modified mathematical formulation for the calculation of axial radiation force was developed to incorporate the effect of ultrasonic attenuation. Axial forces, Fresnel coefficients, average internal attenuation factors and effective internal reflection coefficients were calculated. Thermal and mechanical indices were also computed to address the safety issues in the implementation of acoustic tweezers and were found to be negligible. The results show that the overall distribution of axial forces is barely affected by attenuation. Furthermore, it is found that attenuation actually works against the scattering force and may therefore reinforce the axial trapping force. For a particle size of 180 microm, the maximum trapping force increases from 29.8 x 10(-11) N to 30.3 x 10(-11) N by 1.7% when attenuation is included. In light of these results, it appears that acoustic tweezers may still be feasible beyond the focal point even under the influence of attenuation.     

Inhibition of protein synthesis in intact HeLa cells.

Polysome analysis has proved to be a sensitive probe for the mode of action of inhibitors of protein synthesis in intact HeLa cells. To classify the active compounds as inhibitors of initiation, elongation, or termination, their effects on the cellular polyribosome pattern were compared under three conditions. These conditions tested (i) their direct effect on the polyribosome profile; (ii) their effect on ribosome run-off produced by hypertonicity; and (iii) their effects on recovery from hypertonicity. Using this technique, diacetoxyscirpenol, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, and three alkaloids, harringtonine, isoharringtonine, and homoharringtonine, were found to be inhibitors of initiation. Polysome analysis indicated that in HeLa cells 7.8 x 10(-7) M pactamycin, which inhibited protein synthesis 94%, interfered with elongation as well as initiation under these conditions. Emetine, anisomycin, cycloheximide, and trichodermin each gave polysome patterns consistent with inhibition of elongation. Fusidic acid and aurintricarboxylic acid inhibited incorporation of [(14)C]leucine into intact HeLa cells, but polysome analysis did not localize any specific inhibitory effects to the initiation, elongation, or termination steps of protein synthesis. The use of specific inhibitors of initiation of protein synthesis has indicated that most, if not all, mammalian messenger ribonucleic acids contain a single initiation site.