Receptors for vasoactive intestinal peptide and secretin on guinea pig pancreatic acini

We investigated the abilities of VIP and secretin to occupy receptors and to increase cellular cyclic AMP using dispersed acini from guinea pig pancreas. The dose-inhibition curve for inhibition of binding of 125I-VIP by VIP was broad with detectable inhibition at 0.1 nM VIP, half-maximal inhibition at 2 nM VIP and complete inhibition at 10 microM VIP. Secretin also inhibited binding of 125I-VIP was compatible with two VIP-preferring receptors with one class having a high affinity for VIP (Kd 1.1 nM) and a low affinity for secretin (Kd 5 microM) and the other class having an intermediate affinity for VIP (Kd 470 nM). The dose inhibition curve for inhibition of binding of 125I-secretin by secretin was not broad. Half-maximal inhibition occurred with 7 nM secretin or with 10 microM VIP. Computer analysis was compatible with a single secretin-preferring receptor with a high affinity for secretin (Kd 7 nM) and a low affinity for VIP (Kd 5.9 microM). Comparison of the ability of VIP to increase cyclic AMP with or without the secretin-receptor antagonist, secretin-5-27, demonstrated only occupation of the high affinity VIP-preferring or high affinity secretin-preferring receptors increase cyclic AMP. Our results demonstrate that, in contrast to previous reports, guinea pig pancreatic acini possess 3 classes of receptors that interact with VIP and secretin. The low affinity receptor seen with 125I-VIP is not the same as the secretin-preferring receptor and does not increase cellular cyclic AMP.     

Effects of several calcium antagonists and dipyridamole in the isolated perfused guinea pig heart

Three calcium (Ca) antagonists and dipyridamole were examined in the isolated perfused guinea pig heart at perfusate Ca concentrations of 1.25 and 3.75 mM. The Ca antagonists: FR 7534, nifedipine and D600 produced similar dose-dependent decreases in left ventricular dp/dt and myocardial oxygen consumption (MV?O₂) at both Ca concentrations. However, dose response curves were shifted significantly to the right by increased perfusate Ca requiring six to ten times more Ca antagonist to produce equivalent effects. Dipyridamole produced only slight negative inotropic effects which appeared to be less dependent on external Ca concentration. All four agents significantly increased coronary blood flow at 1.25 mM Ca but not at 3.75 mM Ca. The Ca antagonists decreased heart rate at 3.75 mM Ca whereas dipyridamole had strong negative chronotropic effects at both perfusate Ca concentrations. These experiments provide evidence that FR 7534 acts as a Ca antagonist. In addition, Ca antagonists of different structure had similar effects on the isolated heart distinct from those of dipyridamole.     

Epithelium modulates the potency of vasoactive intestinal peptide in the guinea pig.

The purpose of the study was to evaluate the importance of the epithelium in determining the potency of exogenous vasoactive intestinal peptide (VIP) in inhibiting responses of isolated guinea pig trachea to vagal stimulation. Isolated innervated tracheal preparations (n = 56) were mounted in glass organ baths in Krebs-Henseleit (K-H) solution at 37 degrees C and gassed with 95% O2-5% CO2. The inside of the trachea was separately perfused with K-H solution at 1 ml/min. The vagal nerve trunks were stimulated (20 V, 1-ms pulses, 10-s trains) at low (0.5 Hz) and high frequency (15 Hz) alternately, and the contractile responses were measured as increases in intratracheal pressures. VIP (10(-8)-10(-7) M) inhibited responses to both high- and low-frequency stimulation. VIP was more potent in inhibiting contractions when administered to the outside than the inside surface of the trachea, and disruptionon of the epithelium abolished this difference. The endopeptidase inhibitors phosphoramidon and thiorphan (5 x 10(-6) M) potentiated the action of VIP. These data indicate that the epithelium reduces the efficacy of VIP. We suggest that the epithelium is a site of degradation of VIP by endopeptidase and may also be a diffusion barrier.     

Specificity of receptors to vasoactive intestinal peptide in guinea pig brain.

The specific binding of VIP to guinea pig brain membranes was tested by 1/ the ability of eight VIP and secretin analogs and fragments to inhibit the binding of ¹²⁵I-VIP and 2/ the capacity of the same peptides to influence basal and VIP-stimulated adenylate cyclase activities. Among all peptides tested, only VIP, secretin, [Val⁵] secretin, and [Gln⁹, Asn¹⁵] secretin (5–27) were able to inhibit ¹²⁵I-VIP binding. The adenylate cyclase activity was stimulated by VIP, secretin and [Val⁵] secretin. [Gln⁹, Asn¹⁵] secretin (5–27) although inactive per se was able to inhibit the VIP-stimulated adenylate cyclase activity competitively.     

Hypothermia augments reactive oxygen species detected in the guinea pig isolated perfused heart

Hypothermic perfusion of the heart decreases oxidative phosphorylation and increases NADH. Because O(2) and substrates remain available and respiration (electron transport system, ETS) may become impaired, we examined whether reactive oxygen species (ROS) exist in excess during hypothermic perfusion. A fiberoptic probe was placed on the left ventricular free wall of isolated guinea pig hearts to record intracellular ROS, principally superoxide (O(2)(-).), and an extracellular reactive nitrogen reactant, principally peroxynitrite (ONOO(-)), a product of nitric oxide (NO.) + O(2)(-). Hearts were loaded with dihydroethidium (DHE), which is oxidized by O(2)(-). to ethidium, or were perfused with l-tyrosine, which is oxidized by ONOO(-) to dityrosine (diTyr). Shifts in fluorescence were measured online; diTyr fluorescence was also measured in the coronary effluent. To validate our methods and to examine the source and identity of ROS during cold perfusion, we examined the effects of a superoxide dismutase mimetic Mn(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME), and several agents that impair electron flux through the ETS: menadione, sodium azide (NaN(3)), and 2,3-butanedione monoxime (BDM). Drugs were given before or during cold perfusion. ROS measured by DHE was inversely proportional to the temperature between 37 degrees C and 3 degrees C. We found that perfusion at 17 degrees C increased DHE threefold versus perfusion at 37 degrees C; this was reversed by MnTBAP, but not by l-NAME or BDM, and was markedly augmented by menadione and NaN(3). Perfusion at 17 degrees C also increased myocardial and effluent diTyr (ONOO(-)) by twofold. l-NAME, MnTBAP, or BDM perfused at 37 degrees C before cooling or during 17 degrees C perfusion abrogated, whereas menadione and NaN(3) again enhanced the cold-induced increase in ROS. Our results suggest that hypothermia moderately enhances O(2)(-). generation by mitochondria, whereas O(2)(-). dismutation is markedly slowed. Also, the increase in O(2)(-). during hypothermia reacts with available NO. to produce ONOO(-), and drug-induced O(2)(-). dismutation eliminates the hypothermia-induced increase in O(2)(-).     

The endothelium-dependent and the endothelium-independent vasodilators in the isolated, perfused guinea pig heart.

The endothelium-dependent (acetylcholine, bradykinin, substance P) and the endothelium-independent (gliceryl trinirate, 3-morpholinsydnominine, sodium nitroprusside) vasodilators were studied in the Langendorff-perfused heart of the guinea pig. The involvement of prostanoids and EDRF in the endothelium-dependent responses were assessed by using indomethacin, an inhibitor of cyclooxygenase, and NG-nitro-L-Arginine, an inhibitor of NO synthase. The endothelium-independent agents were used as reference compounds. Both indomethacin and NG-nitro-L-Arginine elevated significantly baseline coronary perfusion pressure, indicating that prostanoids (most likely PGI2 and PGE2) and EDRF modulate the resting tone of the guinea pig coronary circulation. NG-nitro-L-Arginine, but not indomethacin, considerably reduced receptor-stimulated responses. It is concluded that acetylcholine, bradykinin or substance P-induced vasodilation is mediated by EDRF. In contrast, prostanoids do not contribute to endothelium-dependent responses. In addition, short-term tachyphylaxis to bolus injection of gliceryl trinitrate but not of sodium nitroprusside was demonstrated, suggesting that this preparation may be of value for studying nitrate tolerance.     

Effects of platelet-activating factor and thromboxane A2 on isolated perfused guinea pig liver

Lipid mediators, thromboxane A2 (TxA2) and platelet-activating factor (PAF), are potent vasoconstrictors, and have been implicated as mediators of liver diseases, such as ischemic-reperfusion injury. We determined the effects of a TxA2 analogue (U-46619) and PAF on the vascular resistance distribution and liver weight (wt) in isolated guinea pig livers perfused with blood via the portal vein. The sinusoidal pressure was measured by the double occlusion pressure (P(do)), and was used to determine the pre- (R(pre)) and post-sinusoidal (R(post)) resistances. U-46619 and PAF concentration-dependently increased the hepatic total vascular resistance (R(t)). The minimum concentration at which significant vasoconstriction occurs was 0.001 microM for PAF and 0.1 microM for U-46619. Moreover, the concentration of U-46619 required to increase R(t) to the same magnitude is 100 times higher than PAF. Thus, the responsiveness to PAF was greater than that to U-46619. Both agents increased predominantly R(pre) over R(post). U-46619 caused a sustained liver weight loss. In contrast, PAF also caused liver weight loss at lower concentrations, but it produced liver weight gain at higher concentrations (2.5 +/- 0.3 per 10g liver weight at 1 microM PAF), which was caused by substantial post-sinusoidal constriction and increased P(do). In conclusion, both TxA2 and PAF contract predominantly the pre-sinusoidal veins. TxA2 causes liver weight loss, while PAF at high concentrations increases liver weight due to substantial post-sinusoidal constriction in isolated guinea pig livers.     

Receptor binding of guinea pig and pig vasoactive intestinal peptides by rat lung.

Guinea pig vasoactive intestinal peptide (gpVIP) differs from other mammalian VIPs in four of its 28 amino acid residues. In the present study, the gpVIP displaced 125I-labelled pig VIP (pVIP) binding by rat lung membranes with 7.7-fold lower potency than pVIP. Degradation of gpVIP by rat lung membranes, assessed by radioimmunoassay and h.p.l.c., was 1.9-fold greater than that of pVIP. This difference in degradation of the two peptides was not large enough to account for the lower receptor-binding potency of gpVIP. The amino acid residues that distinguish pVIP from gpVIP are likely to contribute to the interaction of VIP with receptors and peptide hydrolases in lung membranes.     

Insulin or glucagon choleresis in the isolated perfused guinea pig liver

Insulin and glucagon choleresis was studied in an in situ, isolated perfused guinea pig liver system. Glucagon caused a small, significant increase in bile salt independent flow (1.83 +/- 0.19 to 2.02 +/- 0.23 microliter g-1 min-1), and dose-related increments over 2-16 micrograms were observed. Insulin alone had no choleretic effect. However, the combination of insulin and glucagon caused a response (1.89 +/- 0.15 to 2.42 +/- 0.19) greater than glucagon alone, and insulin stimulated choleresis when glucagon was present in substimulatory amounts. These observations demonstrate direct effects of glucagon and insulin upon the bile secretory apparatus. Glucagon directly stimulates choleresis, while insulin acts more subtly by potentiation with glucagon.     

The effects of norepinephrine on the release of cyclic AMP by the isolated perfused heart of the guinea pig.

In the isolated perfused heart preparation of the guinea pig, continuous infusions of norepinephrine in doses of 0.06, 0.2 and 0.6 μg/min resulted in a dose-dependent increase in the heart rate, augmentation of the heart contractility measured as increments of dp/dt max. and an increase in the coronary flow. Simultaneous with these changes, a dose-dependent release of substantial quantities of cyclic AMP into the perfusate was observed, and there was a close moment to moment correlation between the increments of dp/dt max. and the increases in the amount of released cyclic AMP produced by norepinephrine. A linear correlation, though less marked than that described above, was also observed between the positive chronotropic and coronary vasodilatatory effects and the increases in the amount of released cyclic AMP, while there was no dynamic correlation between the tissue cyclic AMP level and the augmentation of the mechanical performances of the heart; Increases in the tissue cyclic AMP level were observed only with the highest dose and were transient and a significant decrease was noted with the lowest dose. The decrease was abolished by phentolamine. It is concluded that cyclic AMP exists in different ntracellular compartments in the heart, only one of which -- comprising a small fraction of the total cellular cyclic AMP, -- is functionally-relevant, and that much of this fraction can pass relatively freely across the cell membrane.     

Interaction of porcine vasoactive intestinal peptide with dispersed pancreatic acinar cells from the guinea pig. Structural requirements for effects of vasoactive intestinal peptide and secretin on cellular adenosine 3':5'-monophosphate.

Secretin and vasoactive intestinal peptide (VIP), but not glucagon, stimulate accumulation of cyclic AMP in dispersed guinea pig pancreatic acinar cells. Secretin stimulated cellular accumulation of cyclic AMP by interacting with a single class of high affinity receptors. On the other hand, the dose-response curve for VIP-stimulated cellular cyclic AMP was biphasic and reflected interaction of this peptide with two classes of receptors. Results obtained with synthetic fragments of VIP and secretin indicate that the receptor having a high affinity for VIP has a low affinity for secretin, interacts with, but does not distinguish among, secretin, secretin 5-27 and [6-tyrosine] secretin or among secretin 14-27, VIP 14-28, VIP 15-28, and increases cellular cyclic AMP when occupied by VIP, but not when occupied by secretin, [6-tyrosine] secretin, or secretin 1-14. The receptor having a low affinity for VIP has a high affinity for secretin, interacts with and distinguishes among secretin, secretin 5-27, and [6-tyrosine] secretin, interacts with secretin 14-27 but not with VIP 14-28 or VIP 15-28, and increases cellular cyclic AMP when occupied by VIP, secretin, [6-tyrosine] secretin, or secretin 1-14.     

天然心房肽对实验性心功能与心肌酶的影响

以天然心房肽灌流豚鼠离体工作心脏,药后１分钟心率开始减慢,左室压及其微分开始升高;５分钟后逐渐恢复,至１０分钟恢复正常。体外培养大鼠心肌细胞,培养液中乳酸脱氢酶含量在药后２小时减少至４８．５±６．８ｕ,为药前的４２．７％（Ｐ＜０．０１）,药后８小时仍明显低于药前水平。为心房肽的新药开发提示了新思路。     

血管活性肠肽对支气管上皮细胞趋化迁移的影响及机制

为探讨肺内神经肽在气道损伤修复中的作用 ,采用blind wellBoydenchamber测定原代培养的支气管上皮细胞 (bronchialepithelialcells,BEC)趋化性 ,观察血管活性肠肽 (vasoactiveintestinalpeptide ,VIP)对BEC趋化迁移的影响及其机制 ,并测定经热应激后BEC分泌VIP及表达VIP受体 (vasoactiveintestinalpeptidereceptor,VIPR)的变化。结果显示 :(1)以胰岛素作为趋化因子所建立的BEC趋化性测定方法稳定 ,重现性好 (r =0 970 3,P <0 0 1) ;(2 )VIP (0 0 0 1～ 1μmol/L)均显示剂量依赖性地增强BEC的趋化迁移 ,其效应可被钙调蛋白阻断剂及蛋白激酶C阻断剂有效地抑制 (P <0 0 1) ;(3) 4 2℃、30min热应激后BEC分泌VIP (P <0 0 1)及表达VIPR明显增加 (P <0 0 5 )。实验表明 :肺内神经肽VIP可增强BEC的趋化迁移 ,其细胞内信号转导途径与钙调蛋白及蛋白激酶C有关。而热应激时VIP及VIPR的高表达进一步提示局部微环境的VIP可能是气道上皮损伤修复网络中的重要分子