大鼠内毒素性发热时不同脑区cAMP含量和腺苷酸环化酶活性的变化

给大鼠腹腔注射大肠杆菌内毒素(endotoxin, ET)复制发热模型,观察大鼠ET性发热时不同脑区组织cAMP含量和腺苷酸环化酶(adenylate cyclase, AC)活性的变化。结果发现:大鼠在发热高峰时与对照组比较,丘脑下部cAMP含量明显增加(P<0.01),并与体温变化呈正相关关系(r=0.827);丘脑下部AC活性也显著增强(P<0.001),也与体温变化呈正相关关系(r=0.774)。脑干AC活性显著增强(P<0.05),但与体温变化无正相关关系(r=0.203),cAMP含量也无明显变化。脑皮质cAMP含量和AC活性均无明显变化。以上结果显示:ET可能是通过共同信息物质——内生致热原(EP),以一定方式作用于丘脑下部视前区神经元的细胞膜内AC,使其活化作用于ATP,使ATP分解生成cAMP,从而使局部cAMP含量增加,再通过某种方式使体温调定点上移,导致体温升高。     

Defective leukocyte adenylate cyclase function in hypokalemia

Summary Patients with hypokalemia due to Bartter's syndrome show an increase in adrenergic nervous system function with significantly elevated plasma norepinephrine excretion. Prolonged exposure to neurotransmitters or hormones is known to lead to a down-regulation of target-cell responsiveness. We measured cyclic AMP generation by leukocytes in response to the -adrenergic agonist isoproterenol and to prostaglandin E₁ (PGE₁) in six patients with Bartter's syndrome. As compared to normal controls, the response of cyclic AMP production by leukocytes to stimulation by 1-isoproterenol or PGE₁ was significantly decreased. These results indicate that in Bartter's syndrome and probably in other diseases involving hypokalemia isoproterenol- and PGE₁-sensitive adenylate cyclase activities of leukocytes are reduced. Because the effect of PGE₁ on adenylate cyclase is not mediated through the specific -adrenoceptor, it is possible that a defect in receptor-adenylate cyclase coupling or a more distal post-receptor defect is responsible for the reduction in cyclic AMP production.Abbreviations cyclic AMP adenosine 3:5 - cyclic monophosphate Portions of this work have been presented at the Annual Meeting of the American Society for Clinical Investigation, April 29 to May 2, 1983, Washington, DC (Clin Res 1983; 31:471A)     

Effects of parathyroid hormone and N6,O2′-dibutyryl cyclic AMP on Ca2+ transport across the rabbit distal nephron segments perfused in vitro

Effects on Ca²⁺ transport of parathyroid hormone (PTH) and N⁶,O²-dibutyryl adenosine 3,5-cyclic monophosphate (DB-cAMP) were examined in the rabbit distal nephron segments including the cortical thick ascending limb of Henle's loop (CAL), the connecting tubule (CNT) and the cortical collecting tubule (CCT) by the in vitro perfusion technique. When PTH (10^–8 mol·l^–1) was added to the bath, efflux of Ca²⁺ (pmol·mm^–1·min^–1) was increased from 6.29±1.46 to 7.96±1.66 (P<0.02) in the CAL, and from 8.55±1.30 to 13.73±1.24 (P<0.001) in the CNT, respectively, without changes in influx of Ca²⁺. The effect of PTH on Ca²⁺ transport in the CAL, however, was abolished when phosphate concentration in the medium was reduced from 3.0 to 1.0 mmol·l^–1. When DB-cAMP (10^–3 mol·l^–1) was added to the bath, efflux of Ca²⁺ was also increased from 7.01±0.83 to 9.40±0.82, (P<0.05) in the CAL, and from 13.11±0.89 to 19.74±0.52 (P<0.005) in the CNT, respectively. By contrast, neither PTH nor DB-cAMP affected efflux of Ca²⁺ in the CCT. PTH did not affect the transepithelial voltage either in the CAL or in the CNT. But in the CNT, DB-cAMP decreased the voltage from –14.1 to –9.4 mV. The response of adenylate cyclase activity to PTH in the collagenase treated isolated nephron segments was also examined. Significant increases in adenylase cyclase activity were observed in the CAL as well as in the CNT with 10^–6 mol·l^–1 PTH. These data indicate that PTH stimulates Ca²⁺ transport across the CNT probably via activation of the adenylate cyclase-cyclic AMP system. The hormone may also stimulate Ca²⁺ transport across the CAL in a special condition where plasma phosphate concentration is elevated.     

大鼠无菌性炎症发热时不同脑区cAMP含量和腺苷酸环化酶活性的变化

在大鼠背部皮下注入巴豆油,诱发无菌性炎症(AI)发热,致炎后第一天动物体温上升达高峰,第二天体温开始回降,在第八天体温回降至正常。实验分四组,第一组为对照组,不做致炎处理,第二组为发热高峰组,第三组为发热恢复组,观察大鼠AI发热高峰期及恢复期时不同脑区cAMP含量和腺苷酸环化酶(AC)活性的变化。第四组为发热恢复正常组.观察致炎后第一天至第八天动物体温从高峰障至正常的变化曲线,不做脑组织取材。结果发现:大鼠在发热高峰期与对照组比较,丘脑下部cAMP含量明显增加(P<0.01),并与体温变化呈正相关关系(r=0.893);AC活性显著增强(P<0.001),也与体温变化呈正相关关系(r=0.824),脑皮质、脑干cAMP含量与AC活性均无明显变化。发热恢复组与对照组比较:丘脑下部cAMP含量明显增加(P<0.05),丘脑下部、脑干AC活性均显著增强(P<0.05),而脑皮质cAMP含量和AC活性均无明显变化。作者推论:大鼠AI发热很可能是由于丘脑下部AC活性增强使局部cAMP含量增多所致。     

Mutations affecting cyclic phosphodiesterases and adenylate cyclase in Neurospora

Summary Two regulatory mutants in orthophosphateregulated cyclic phosphodiesterase (CPDase), cpd-3 and cpd-4, were isolated and mapped proximal to arg-1 on L.G. IC and distal to urg-12 on L.G. IIR, respectively. cpd-3 showed short aerial hyphae with dense formation of conidia. The morphology was very similar to that of cr-1, cpd-3 and cr-1 had reduced levels of cyclic 3,5-AMP, adenylate cyclase and cPDases (CPDase I, II and III in low phosphate condition) but had elevated levels of cyclic 3,5-GMP. Although cr-1 showed an enhanced level and enhanced activation of heat activated cyclic phosphodiesterase (ha-PDE), this enzyme was not activated in cpd-3. cpd-4, nuc-1 and nuc-2 produced neither CPDase I, II, III, alkaline phosphatase nor ribonuclease N1 in low phosphate media. These mutants did not produce aerial hypha or conidium when grown in low phosphate liquid medium. Activation of ha-PDE occurred in cpd-4, but not in nuc-1 and nuc-2.     

Ionic mechanism of inotropic effect of prostaglandin E1 on frog atrial muscle

Summary Prostaglandin E₁ increases the amplitude and duration of cardiac action potential and exerts a positive inotropic effect on the frog heart. In voltage-clamp experiments, prostaglandin E₁ increases both, the slow inward current and the delayed outward as well as the two components of the heart contraction. These results imply that calcium influx and the release of intracellular calcium are involved in the inotropic action of prostaglandin E₁.     

The effect of prostaglandin E2 and ADH on diffusional water permeability in the collecting duct of an isolated rat papilla

The effect of prostaglandin on diffusional water permeability has been studied in collecting ducts in an isolated rat papilla. PGE₂ increased water permeability. The effect was significant at a concentration of 10^–8 mol l^–1 and was maximal with a concentration of 10^–6 mol l^–1. The maximal increment of 0.94±0.10 (SEM) m s^–1 was approximately half that produced by maximal stimulation with antidiuretic hormone (2.18±0.12 m s^–1).A concentration of 10^–8 mol l^–1 produced an increase in basal water permeability and 25 unit ml^–1 ADH, which without PGE₂ present gave a similar increase, had no incremental effect. ADH 100 unit ml^–1 increased permeability to a value similar to that observed in the absence of PGE₂. Thus PGE₂ and ADH both increase water permeability but the increments are not additive.Indomethacin in a concentration that inhibited prostaglandin production altered the response of the collecting duct to ADH. The dose response curve was shifted to the left and the maximal increase in water permeability and the lowest dose at which a response occurred took place at concentrations less than 1/2 those required in its absence.Prostaglandins influence the action of ADH and it is likely that in life they regulate and modulate the change in water permeability induced by anti-diuretic hormone.     

The effect of vasoactive intestinal polypeptide (VIP) on forskolin stimulated adenylate cyclase in the caudate-putamen of the rat

Summary Vasoactive intestinal polypeptide (VIP) was incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen (CP) tissue of the rat in order to examine the effect of the peptide on forskolin-activated adenylate cyclase in vitro. Forskolin induced an enhancement of cyclic AMP formation that was mediated by an effect on catalytic subunit and stimulatory guanine nucleotide regulatory protein (N_s). In our preparation, VIP did not influence basal adenylate cyclase activity or the stimulation by dopamine and sodium fluoride but, in the absence of guanylylimidodiphosphate (guanosine 5-(,y-imido)-triphosphate) VIP inhibited the forskolin-stimulation of the enzyme in a noncompetitive manner. Met-encephalin, acting on a D-2 receptor-coupled putative inhibitory guanine nucleotide regulatory protein (N_i), inhibited the adenylate cyclase activity stimulated by forskolin to a slightly greater extent than VIP. When assayed together, these inhibition effects were additive, implying that the peptide receptors are not identical. The N_i — antagonist, MnCl₂ completely blocked the inhibition of met-encephalin but had no significant effect on VIP-induced inhibition. In addition, pertussis toxin did not influence the effect of VIP on forskolin-stimulation in contrast to cholera toxin which did antagonize the VIP effect via the stimulatory guanine nucleotide regulatory protein (N_s). Furthermore, specific D-1 and D-2 dopaminergic receptor antagonists (+)-flupentixol and spiperone had no effect on VIP-modulated forskolin-stimulated adenylate cyclase activity. These results suggest that the neuromodulatory effect of VIP is mediated by a N_s distinct from those involved in several adenylate cyclase pools sensitive to stimulation by dopamine and VIP in the rat striatum.     

Effect of glucagon on adenylate cyclase activity and acid production of isolated human parietal cells

Summary The direct effect of glucagon on human parietal cell function in vitro was tested by measuring adenylate cyclase (AC) activity and H⁺ production in homogenates of human gastric mucosa obtained during surgery or at biopsy. Cells isolated from mucosa obtained during surgery showed an increase in AC with histamine and glucagon. In parietal cell enriched fractions (75%) glucagon and histamine stimulated AC much more effectively than in parietal cell depleted fractions (15% and 7%). In contrast, glucagon did not affect basal or histamine stimulated¹⁴C amino pyrine uptake.In homogenates of mucosal biopsy specimens 2×10^–7 mol/l glucagon enhanced AC activity by 76% (corpus) and 20% (antrum). In the same homogenates 10^–4 mol/l histamine caused a stimulation by 161% (corpus) and 38% (antrum). In fundic biopsy specimens glucagon displayed a biphasic concentration response curve with an increase at 10^–10 mol/l (46% above basal AC activity) and a maximum at 2×10^–7 mol/l (97%). Histamine elicited the maximal response (192%) at 10^–3 mol/l. Increasing histamine and glucagon concentrations caused additive stimulation of AC. Ranitidine did not change AC in response to glucagon but abolished the effect of histamine. Data suggest that the glucagon action is mediated by separate (glucagon?) receptors. As H⁺ production was not affected by glucagon, the coexistence of two AC systems in the human parietal cell is postulated: One that is activated via histamine H₂-receptors and which stimulates H⁺ production; another that is activated by glucagon and is directed towards other, possibly metabolic effects.Abbreviations AC Adenylate cyclase - cAMP Cyclic adenosine monophosphate - ATP Adenosine triphosphate Supported by grants from the Deutsche Forschungsgemeinschaft Mi 170/2-2Dedicated to Prof. Dr. F. Krück on the occasion of his 65th birthday     

The gastric cytoprotective action of adenosine and prostaglandin E2 in rabbits

The direct protective action of adenosine and prostaglandin E₂ (PGE₂) was examined in an isolated gastric gland preparation in rabbits. Ethanol, (8%, v/v) incubation markedly increased the release of lactate dehydrogenase (LDH) and number of non-viable glands in the preparation. Both effects were prevented by PGE₂ preincubation in a concentration (10^–6, 1.4×10^–5 or 2.8×10^–5 M)-dependent manner. The protective action was smaller in adenosine-treated groups, and yet the highest concentration (10^–4 M) of the compound also significantly inhibited the cytotoxic effects of ethanol. These findings indicate that both adenosine and PGE₂ possess cytoprotective action on gastric glands in rabbits, but the former compound exerts its action beyond physiological concentrations. It is concluded that endogenous PGE₂, but not adenosine may act as an ulcer modulator in the stomach.     

Gene expression of the human prostaglandin E receptor EP4 subtype: differential regulation in monocytoid and lymphoid lineage cells by phorbol ester

We isolated a cDNA clone encoding the human prostaglandin (PG) E receptor EP₄ subtype and examined the gene expression in human blood cells. Northern blot analysis revealed that the EP₄ gene is expressed at a high level in peripheral blood mononuclear cells, and at lower levels in cultured human blood cell lines, THP-1 and U937 (monocytoid cell lines), MOLT-4 and Jurkat (T-cell lines), and Raji (B-cell line). To examine regulation of the EP₄ gene expression in the immune system, we studied the effects of phorbol 12-myristate 13-acetate (PMA) on these cell lines. Gene expression was upregulated in THP-1, U937, and Raji cells by PMA, and was downregulated in MOLT-4 and Jurkat cells. In THP-1 cells the effects of PMA were further analyzed, and the upregulation of the EP₄ gene was shown to be followed by an increase in PGE₂ binding sites and in PGE₂-induced cAMP accumulation. In the striking contrast, other PGE receptor subtypes (EP₁, EP₂ and EP₃) and other prostanoid receptors (IP and DP) were shown not to be upregulated by PMA. Therefore, this is the first demonstration of a highly specific upregulation of the EP₄ subtype in THP-1 cells treated with PMA, suggesting the importance of the EP₄ subtype in the immune system. In the present study we also clarified that EP₄ gene expression is regulated differently among human monocytoid and lymphoid lineage cells, thus leading to the better understanding of the regulatory mechanisms for the human EP₄ gene expression in the immune system.Abbreviations PG Prostaglandin - PMA Phorbol 12-myristate 13-acetate - PBMC Peripheral blood mononuclear cells     

Inhibition of prostaglandin synthesis reduces cyclic AMP levels and inhibits chondrogenesis in cultured chick limb mesenchyme

The present study investigated effects of inhibiting the synthesis of prostaglandins (PGs) on cyclic AMP concentrations and chondrogenesis in cultured chick limb mesenchyme. Indomethacin produced concentration-dependent inhibition of both PGE₂ synthesis and chondrogenesis over a concentration range of 50--200 M. Half maximal inhibition of PGE₂ was achieved with 50 M concentrations of the drug which also produced visibly reduced amounts of cartilage matrix in cell cultures as evaluated by Alcian green staining on day 6 of culture. The inhibitory effects of indomethacin on chondrogenesis were largely reversed by addition of 1 mM dibutyryl cAMP, indicating that cells could still respond to cyclic AMP stimulation. Endogenous levels of cyclic AMP, which increased by 6 fold during the six days of culture in control cells, did not increase significantly from dissociated cells at the time of plating (day 0) in indomethacin-treated cultures. The results indicate that inhibition of the prechondrogenic rise in PGE₂ concentrations in limb mesenchyme prevents the increase in cyclic AMP levels which occur during this same period resulting in inhibition of chondrogenesis. The data provide further support for the hypothesis that PGE₂, through its effects on the adenylate cyclase-cAMP system, plays an important role in the differentiation of cartilage.     

Cyclic AMP dependent,constitutive thermotolerance in the adenylate cyclase-deficient cr-1 (crisp) mutant of Neurospora crassa

Summary We investigated the heat shock response of the adenylate cyclase deficient mutant cr-1 (crisp) of Neurospora crassa. This strain was observed to be much more resistant to a lethal temperature of 50 °C than the wild type. This constitutive thermotolerance was absent in cr-1 conidiospores raised on cyclic AMP (cAMP, 2.5 mM) supplemented solid medium, but was partially restored when the conidiospores were germinated at 30 °C, a temperature which fails to induce thermotolerance in the wild-type strain. Two other crisp-like Neurospora mutants, cr-2 and cr-3 which, in contrast to cr-1, contain normal levels of cAMP, did not exhibit the thermotolerance phenomenon observed for cr-1. A cr-1, pe, fl (crisp-microconidial) strain also lacked the ability to tolerate a lethal heat treatment. Our results demonstrate that the adenylate cyclase deficient cr-1 mutant of Neu-rospora crassa expresses a constitutive thermotolerant phenotype as a consequence of its primary genetic defect: low levels of cAMP.     

Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis

A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.     

Circulation parameters during intravenous and intra-arterial administration of increasing doses of prostaglandin E1 in healthy subjects

Summary Increasing doses of prostaglandin E₁ given intravenously do not influence the hemodynamic parameters up to a dosage of 1500 ng/min. Intra-arterial administration of a mixture of nucleotides and nucleosides in therapeutic doses induces an increase in the blood flow volume at rest of up to 400%. With dosages of 20 ng/min and more, intra-arterial administration of PGE₁ induces only a slight increase in the blood flow volume of the extremity under treatment. The therapeutic effects of the substance, therefore, are probably more due to other mechanisms of action than to hemodynamic effects.Abbreviations ATP adenosintriphosphate - HF heart rate - HZV cardiac output - NNG mixture of nucleotides and nucleosides - PGE₁ prostaglandin E₁ - VOL blood flow volume Dedicated to Professor Hans J. Dengler on the occasion of his 60th birthday     

Comparison of the action of prostaglandin with endotoxin on thermoregulatory response thresholds

Prostaglandin E₂ (PGE₂) and lipopolysaccharide (LPS) derived fromE. coli were injected into the lateral cerebral ventricle of rabbits at 30° C ambient temperature. The threshold core temperatures for ear cutaneous vasoconstriction. (Th _v) and shivering (Th _sh) were determined by whole-body cooling with an intestinal thermode. Each threshold, as determined at the plateau phase of LPS fever and PGE₂ hyperthermia respectively, were compared with the control values before LPS and PGE₂ injection.Th_sh was not changed by the injection of LPS, whileTh _v was increased. After PGE₂ injection bothTh _sh andTh _v were increased in comparison to their control levels. These changes paralleled the elevation of core temperature.The present study does not exclude prostaglandins as humoral mediators involved in some of the central processes generating fever, but suggest at the same time that there are additional properties of LPS fever for which prostaglandins do not account.