Vasoactive intestinal peptide augmentation of cholecystokinin-8-stimulated pepsinogen secretion: evidence for dual modulation of chief cell function

Pepsinogen (PPG) secretion from chief cells (CC) is dually modulated by adenosine 3':5'-monophosphate (cAMP) and calcium second messenger systems. Vasoactive intestinal peptide (VIP) stimulates cellular function by elevating intracellular levels of cAMP. In contrast, cholecystokinin-8 (CCK-8) acts by producing a rise in intracellular calcium concentration. Consequently, it was the purpose of this study to test whether VIP (acting through cAMP-mediated systems) could augment CCK-8 (acting through calcium-dependent systems)-stimulated PPG secretion. Collagenase dispersed rabbit isolated gastric glands (IGG) were incubated alone (unstimulated) or with secretagogues for 30 min. VIP in graded doses of 10(-11) to 10(-7) M was used alone or in combination with CCK-8 (10(-9) M). PPG levels were determined using an assay based on pepsin hydrolysis of [14C]methemoglobin. Results are expressed as percentage of total pepsinogen (within the IGG) secreted above unstimulated levels. VIP alone (10(-11) to 10(-7)) or CCK-8 alone (10(-9)) did not significantly stimulate PPG secretion (P greater than 0.05). The combination of CCK-8 (10(-9) M) plus VIP (10(-7) M) significantly stimulated PPG secretion above unstimulated levels (P less than 0.05). Thus, the combination of VIP and CCK-8 produced greater PPG secretion than either secretagogue alone. These data support the hypothesis that secretagogues acting through either cAMP or calcium-mediated systems contribute to the regulation of PPG secretion from CC and that the two second messenger systems act in concert achieving at least additive effects.     

Age-dependent stimulation of neonatal insulin release and inositol phosphate accumulation by CCK-8 and carbachol

Development of a robust insulin secretory response to glucose occurs during the early neonatal period. To determine if neuroendocrine agents play a role during this time, we studied the effects of selected peptides and neurotransmitters on insulin release and polyphosphoinositide metabolism in islets isolated from 1- and 3-day neonatal rats. Vasoactive intestinal peptide had no effect on glucose-stimulated release in either islet population. In contrast, sulfated cholecystokinin octapeptide (CCK-8) significantly enhanced glucose-induced insulin release in both islet groups. One-day islets were stimulated only by a concentration of 300 nM, whereas 3-day islets were responsive at 3 nM. Similar to CCK-8, there were clear differences in responses to carbachol between 1- and 3-day islets. One-day islets required a concentration of 200 microM for insulin release to be significantly greater than with glucose alone; 3-day islet insulin release was significant at 2 microM carbachol. Both agonists stimulated inositol phosphate accumulation in 3-day islets, but only CCK-8 caused a significant increase over glucose-induced levels in 1-day islets. These results indicate that islet responsiveness to CCK-8 and carbachol develops in parallel during the early neonatal period. This development may be linked to the maturation of a critical step of stimulus-secretion coupling through which these agents act.     

Endotoxin, epinephrine, glucagon, insulin and calcium ionophore A23187 modulation of pyruvate kinase activity in cultured rat hepatocytes

Altered glucose metabolism is one of the commonly observed sequelae of sepsis and septic shock. The present investigation was undertaken to determine the role of endotoxin (ET) upon hepatocyte glucoregulation, by measuring the activity of pyruvate kinase (PK), a key glycolytic enzyme. Hepatocytes were exposed to endotoxin concentrations known to occur in vivo during sepsis, i.e., from 1 X 10(-14) to 1 X 10(-8) g/ml. The alteration of the enzyme activities after addition of epinephrine, glucagon, insulin and calcium ionophore A23187 with and without ET preincubation were also examined. ET alone decreased the PK activity by 12% at all concentrations tested. The basal inhibition of the enzyme caused by epinephrine (-48%) was partially blocked by ET preincubation above 1 X 10(-10) g/ml. There were no ET-(glucagon, calcium ionophore, insulin) interaction. These in vitro results do not support pyruvate kinase as a site of hepatic enzyme regulation defect in endotoxaemia.     

Modulatory effect of glucose, amino acids, and secretin on CCK-8-induced somatostatin and pancreatic polypeptide release in dogs

Protein- and fat-rich test meals elicit a strong stimulatory effect on postprandial somatostatin (SLI) and pancreatic polypeptide (PP) release, whereas carbohydrate-rich meals rather attenuate the response of both hormones. Since there is evidence that intestinal hormones might contribute to the postprandial SLI and PP response, it was the aim of the present study to determine in dogs the effect of low-dose cholecystokinin octapeptide (CCK-8) on basal hormone levels and also during a background infusion of amino acids or glucose. In a group of six conscious dogs, sulfated CCK-8 was infused intravenously (i.v.) via a hindleg vein at stepwise increasing infusion rates of 10, 30, and 50 pmol X kg-1 X h. The infusion of CCK was applied during a background infusion of saline (2 ml/min), glucose (0.2 g/min), or an amino acid mixture (8.5%, 2 ml/min). CCK-8 had no effect on plasma insulin and glucagon levels under all experimental conditions. Plasma SLI levels were significantly stimulated by all doses of CCK. This stimulatory effect was similar during background infusions of either saline, glucose, or amino acids, respectively. Pancreatic polypeptide (PP) levels rose 200-300 pg/ml during CCK plus saline. This was slightly attenuated by glucose. During CCK plus amino acids, the PP response was augmented to 600-800 pg/ml. Since secretin is also released after the ingestion of a meal and intraduodenal acidification is a potent stimulus not only of secretin but also of gastric and pancreatic SLI release, the effect of secretin was examined additionally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium uptake, respiration, and ultrastructure of sperm exposed to ionophore A23187

Calcium accumulation by ram sperm in the presence of 3.0 microM ionophore A23187 increased greatly when the pH of the medium was raised from 7.5 to 8.5. The increase in calcium uptake was remarkable between pH 7.5 and 8.0 and was paralleled by increased oxygen consumption. There was extensive vesiculation of the plasma membrane and membranes of the acrosome and postnuclear cap of ram sperm incubated with the ionophore and calcium at pH 8.0. The mitochondria of the midpiece contained pale and expanded cristae similar to ATP-deprived mitochondria in the "condensed" configuration. Such changes were not observed in ram sperm incubated under similar conditions at pH 7.0. The ionophore-induced calcium and oxygen uptake of cauda and caput epididymal boar sperm also increased with increased pH, but the effect commenced at a lower pH. Control cauda epididymal boar sperm (i.e., without ionophore) had higher oxygen uptake, glucose breakdown, and lactic acid production than caput sperm. The influence of pH on the operation of the membrane calcium pumps between ram boar sperm indicates species differences.     

Pulse oximeter probes. A comparison between finger, nose, ear and forehead probes under conditions of poor perfusion

The performances of 10 pulse oximeters using finger probes were compared with the same pulse oximeters using alternative probes (eight finger probes, two nose probes and a forehead probe) in poorly perfused patients. All readings were then compared with directly measured arterial blood oxygen saturations. The mean difference (bias, 'accuracy'), standard deviation (precision) and 'drop out' rate for each pulse oximeter combination was determined. An overall ranking of performance of each pulse oximeter was calculated using five criteria (accuracy, precision, number of readings within 3% of standard, percentage of readings given within 3% of standard, expected overread limit in 95% of cases). Nose and forehead probes performed poorly. Some ear probes performed well compared to some finger probes, but the overall performance of probes in other sites compared to finger probes was worse, (p = 0.05). Two of eight ear probes and no nose or forehead probes would be expected to be within 4% of the reference value in 95% of readings. The use of finger probes rather than probes in other sites is recommended in the patient with poor peripheral perfusion.     

Glucose-dependent modulation of insulin secretion and intracellular calcium ions by GKA50, a glucokinase activator

Because glucokinase is a metabolic sensor involved in the regulated release of insulin, we have investigated the acute actions of novel glucokinase activator compound 50 (GKA50) on islet function. Insulin secretion was determined by enzyme-linked immunosorbent assay, and microfluorimetry with fura-2 was used to examine intracellular Ca(2+) homeostasis ([Ca(2+)](i)) in isolated mouse, rat, and human islets of Langerhans and in the MIN6 insulin-secreting mouse cell line. In rodent islets and MIN6 cells, 1 micromol/l GKA50 was found to stimulate insulin secretion and raise [Ca(2+)](i) in the presence of glucose (2-10 mmol/l). Similar effects on insulin release were also seen in isolated human islets. GKA50 (1 micromol/l) caused a leftward shift in the glucose-concentration response profiles, and the half-maximal effective concentration (EC(50)) values for glucose were shifted by 3 mmol/l in rat islets and approximately 10 mmol/l in MIN6 cells. There was no significant effect of GKA50 on the maximal rates of glucose-stimulated insulin secretion. In the absence of glucose, GKA50 failed to elevate [Ca(2+)](i) (1 micromol/l GKA50) or to stimulate insulin release (30 nmol/l-10 micromol/l GKA50). At 5 mmol/l glucose, the EC(50) for GKA50 in MIN6 cells was approximately 0.3 micromol/l. Inhibition of glucokinase with mannoheptulose or 5-thioglucose selectively inhibited the action of GKA50 on insulin release but not the effects of tolbutamide. Similarly, 3-methoxyglucose prevented GKA50-induced rises in [Ca(2+)](i) but not the actions of tolbutamide. Finally, the ATP-sensitive K(+) channel agonist diazoxide (200 micromol/l) inhibited GKA50-induced insulin release and its elevation of [Ca(2+)](i.) We show that GKA50 is a glucose-like activator of beta-cell metabolism in rodent and human islets and a Ca(2+)-dependent modulator of insulin secretion.     

Responses of fetal rat bone cells and bone organ cultures to the ionophore,A23187

The ionophore A23187 produced a rapid transient increase in the rate of calcium uptake by isolated fetal rat bone cells. There was no effect on calcium efflux or total cellular calcium. The magnitude of the effect on influx was amplified when the cell were incubated at 4°C. Cellular metabolic functions and resorption of cultured fetal rat bones (release of⁴⁵Ca from pre-labeled long bone) were affected by A23187 in a biphasic manner: cell cyclic AMP (cAMP) was increased by 0.1 and 0.3 g/ml of the ionophore, whereas 10 g/ml was either ineffective or lowered the cAMP levels. The high A23187 concentration abolished the stimulatory effects of parathyroid hormone and methylisobutylxanthine. Concentrations of 0.1 and 0.3 g/ml A23187 stimulated bone resorption. The effect was abolished by calcitonin. Ionophore concentrations above 1 g/ml produced less bone resorption. These higher concentrations antagonized the bone-resorbing effect of parathyroid hormone and 1,25-dihydroxyvitamin D₃. A23187 at 5 and 10 g/ml decreased bone cell lactate and ATP. Thus at low concentrations, A23187 produced effects on bone similar to those of parathyroid hormone, suggesting that calcium is the primary initiator of PTH-induced bone resorption. At the higher concentrations A23187 may have a general inhibitory effect on cell metabolism.     

A modified apparatus for dual, sterilized, isolated perfusion of the rat liver

The isolated perfused rat liver (IPRL) has proven to be a useful model for the study of physiology and pathology of the liver. For research in nonparenchymal cell (NPC) function that includes measurement of cytokine production (eg, TNF), it is necessary to have a sterilized perfusion system. We have modified the IPRL apparatus so as to be able to perform sterile perfusions of two livers simultaneously. The perfusion apparatus is a recirculating closed system in which the oxygenator is a plastic container separated into two chambers by a fenestrated plastic wall. A disposable macropore filter functions as both a bubble trap and perfusate filter. The sterilization process is done by immersing the various components in Benz-All solution. The tubing is disinfected by irrigation with 10% Clorox followed by 0.9% sodium chloride solution. The perfusate used is filter-sterilized Krebs buffer solution containing 0.5 g Mandol/250 mL perfusate. Not only can two organs be conveniently perfused simultaneously, but the entire system can be reliably sterilized for up to 20 consecutive perfusions. Bile production is higher and more stable with less leakage of intracellular enzymes. Many of the components are disposable and can be altered to suit the needs of a particular experiment.     

己酮可可碱、孕酮和A23187对人精子甘露糖受体表达的影响

人精子经ＢＷＷ获能５～６小时后，分别用己酮可可碱（ＰＦ）、孕酮（Ｐ）和Ａ２３１８７三种已知的获能及（或）顶体反应促进剂处理１小时，对实验和对照组精子用异硫氰酸荧光素标记的甘露糖化牛血清白蛋白（Ｍ ＦＩＴＣ ＢＳＡ）作探针标记精子甘露糖受体（ＭＲ）。结果表明，获能促进剂ＰＦ并不促进ＭＲ表达而Ｐ和Ａ２３１８７则有显著促进ＭＲ表达的作用。Ｐ对ＭＲＩＩ型的促表达作用略强于对ＩＩ型，而Ａ２３１８７则对ＩＩ型的促进显著强于Ｉ型。认为ＭＲ的表达是一种依赖于钙离子的细胞生物学过程，并与顶体反应有密切关系。推测ＭＲ可能具有介导精子与卵膜融合的作用。     

Evidence for the synthesis and secretion of a CBG-like serpin by human cumulus oophorus and fallopian tubes

Summary.  The acrosome reaction (AR)-inducing effect of follicular cells, like that of the cumulus oophorus and granulosa cells, has been described previously. In addition to the well known steroid secreting activity of cumulus cells, the results obtained here demonstrate the secretion of a corticosteroid-binding globulin (CBG)-like protein. An AR-inducing effect was shown with the culture medium of human cumulus oophorus. This effect could be eliminated by treating the sample with monoclonal antibodies against CBG. Moreover, Western blotting after SDS-PAGE of the culture medium strongly indicates that human cumulus cells actively express and secrete a CBG-like protein. This might give an indication as to the origin of the acrosome reaction-inducing substance found in follicular fluid. Furthermore, AR-inducing activity and the elimination of this activity by antibodies against CBG was shown for oviductal fluid. With immunohistochemical techniques the CBG-like protein was localized in the epithelial lining of the fallopian tubes, giving possible evidence for the involvement of this molecule in fallopian tube function.     

Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: the effects of heparin and calcium ionophore A23187

In this study, we investigated the effect of heparin and calcium ionophore A23187 on in vitro induction of buffalo sperm acrosome reaction (AR). Two methods for detection of the AR and viability were employed. Fluorescein isothiocyanate-conjugated Arachis hypogea agglutinin (FITC-PNA) was used as a vital stain in combination with ethidium homodimer-1 (EthD-1) to determine the acrosome status of viable spermatozoa. In another experiment, trypan blue replaced EthD-1 to differentiate live and dead spermatozoa having undergone AR. The results from the two methods were significantly correlated (r > 0.9). Four different staining patterns were found in both methods. The FITC-PNA intensely labels the acrosome region of acrosome-intact spermatozoa. EthD-1 and trypan blue stained red and blue at the post-acrosomal region of dead spermatozoa, respectively. Spermatozoa incubated with heparin showed a significant increase ( p < 0.05) in the percentage of live acrosome-reacted sperm after 30 min incubation period. This trend continued and was significantly different over the entire incubation period when compared with the control group at the same interval. In the ionophore-treated group, the proportion of changes in live acrosome-intact and live acrosome-reacted spermatozoa was statistically significantly different ( p < 0.001) when compared with those treated with heparin at the same interval. The AR occurred sooner and to a greater extent when incubated with the ionophore but at 5 h of incubation the percentage of false acrosomal reaction was significantly higher than those in the control and heparin-treated groups. The results in this study indicated that the in vitro induction of AR by heparin and calcium ionophore evaluated by both methods could be used to assess sperm fertilizing capacity for in vitro fertilization of this species.     

钙离子载体A23187联合卵细胞胞质内单精子注射治疗圆头精子症2例并文献复习

目的:探讨圆头精子症的病因、圆头精子的受精能力,以及人工激活技术在圆头精子症患者卵细胞胞质内单精子注射(ICSI)治疗中的应用价值。方法:回顾性分析2例圆头精子症ICSI治疗病例,患者配偶的卵子经ICSI处理后,随机分为2组,其中1组卵子接受钙离子载体A23187激活处理,另外1组不接受任何处理。结合国内外文献对圆头精子症的发病原因、受精能力和治疗方案进行复习和讨论。结果:A23187激活处理组均获得优质胚胎,而常规处理对照组的卵子则未受精、未卵裂、无优质胚胎、无囊胚形成。2例患者均移植A23187激活组的优质胚胎,获得妊娠并分娩健康婴儿。结论:A23187可以用于圆头精子症患者的ICSI治疗,并获得较好的临床结局,但还应密切关注A23187的安全性问题。     

Evidence for opioid modulation and generation of prostaglandins in sulphur dioxide (SO)2-induced bronchoconstriction.

BACKGROUND: Inhalation of sulphur dioxide (SO2) provokes bronchoconstriction in asthmatic subjects. Cholinergic mechanisms contribute, but other mechanisms remain undefined. The effect of morphine, an opioid agonist, on the cholinergic component of SO2-induced bronchoconstriction was investigated, and the effect of indomethacin, a cyclooxygenase inhibitor, on SO2-induced bronchoconstriction and tachyphylaxis was studied. METHODS: In the first study 16 asthmatic subjects inhaled either ipratropium bromide or placebo 60 minutes before an SO2 challenge on days 1 and 2. On day 3 an SO2 challenge was performed immediately after intravenous morphine. In the second study 15 asthmatic subjects took either placebo or indomethacin for three days before each study day when two SO2 challenges were performed 30 minutes apart. The response was measured as the cumulative dose causing a 35% fall in specific airways conductance (sGaw; PDsGaw35). RESULTS: Ipratropium bromide significantly inhibited SO2 responsiveness, reducing PDsGaw35 by 0.89 (95% CI 0.46 to 1.31) doubling doses. This effect persisted after correction for bronchodilatation induced by ipratropium bromide. The effect of ipratropium bromide and morphine on SO2 responsiveness also correlated (r2 = 0.71). In the second study SO2 tachyphylaxis developed with PDsGaw35 on repeated testing, being reduced by 0.62 (95% CI 0.17 to 1.07) doubling doses. Indomethacin attenuated baseline SO2 responsiveness, increasing PDsGaw35 by 0.5 (95% CI 0.06 to 0.93) doubling doses. CONCLUSIONS: These results suggest that opioids modulate the cholinergic component of SO2 responsiveness and that cyclooxygenase products contribute to the immediate response to SO2.     

Urological applications of human papillomavirus typing using deoxyribonucleic acid probes for the diagnosis and treatment of genital condyloma

The use of deoxyribonucleic acid probes to examine the type of the human papillomavirus genome found in penile lesions is described at a technical level and in a clinical application. At least 40 different types of human papillomavirus have been identified and these types vary not only in their deoxyribonucleic acid base sequences but also in their clinical manifestations. Although deoxyribonucleic acid probes currently have a role only in a research setting, this study delineates the possible role of this technology in a clinical setting to detect subclinical intraurethral human papillomavirus. The results have widespread implications regarding the treatment of condyloma and the associated cervical dysplasia. In this series 25 grossly visible lesions were typed and 85 per cent contained human papillomavirus types 6 and 11. In contrast, microscopic lesions identified in the male partners of women with cervical dysplasia were shown to contain human papillomavirus types 16, 18 or 31 in 60 per cent of the cases. In addition, urethral brushings were obtained and were positive for human papillomavirus in 50 per cent of the cases despite normal urethroscopy. Human papillomavirus types 16, 18 or 31 accounted for 70 per cent of the positive urethral brushings.     

Evidence for tissue-directed immune responses: analysis of CD4- and CD8-dependent alloimmunity

BACKGROUND: Transplant rejection has generally been considered a CD4 T-cell-dependent immune process. CD4-independent, CD8 T-cell rejection pathways have recently gained attention because of their relative resistance to immunosuppression. In the current study, the role of the allograft tissue in activation of these distinct pathways was examined by comparing host-immune responses with allogeneic pancreatic islets or hepatocytes transplanted across the same genetic disparity. METHODS: To compare activation of CD4-dependent versus CD8-dependent alloimmunity, islets or hepatocytes retrieved from FVB/N (H-2) mice were transplanted into CD8 or CD4 T-cell-reconstituted severe combined immunodeficiency mice, CD4 or CD8 knockout (KO) mice, and anti-CD4 monoclonal antibody (mAb) or anti-CD8 mAb treated C57BL/6 mice (all H-2). The ability to immunomodulate CD4-dependent allograft rejection (in CD8 KO mice) was examined in the context of several mechanistically distinct immunotherapeutic strategies, including anti-CD4 mAb, donor-specific transfusion and anti-CD154 mAb, and anti-lymphocyte function-associated antigen-1 mAb. RESULTS: The studies demonstrate that, whereas hepatocytes evoke alloreactive CD4-dependent and (CD4-independent) CD8 T-cell immune responses, allogeneic islets only activate CD4-dependent immune pathways. CD4-dependent host-immune responses initiated by pancreatic islet allografts were readily suppressed by a variety of short-term immunotherapies, whereas hepatocyte-initiated CD4-dependent alloimmune responses were not. CONCLUSIONS: These results demonstrate that immune characteristics of the specific allograft tissue uniquely influence the pattern of host immune responses such that the propensity to activate CD4- or CD8-dependent alloimmune responses can be distinguished. Furthermore, CD4-dependent immune responses activated by different tissues from the same donor strain are distinguished by their susceptibility to specific immunotherapy.     

Evidence for the involvement of host-derived OKT8-positive T cells in the rejection of T-depleted, HLA-identical bone marrow grafts

The recent introduction of a variety of techniques for removing T cells from bone marrow grafts has reduced the incidence of graft-versus-host disease (GVHD)-associated morbidity and mortality. Whether this advance will be translated into improved patient survival is unclear at present, mainly because these procedures increase the risk of graft failure. Since 1983 we have transplanted 25 consecutive leukemia patients with HLA-identical sibling grafts purged of T cells by a single incubation with the monoclonal antibody Campath-1 and donor complement. This approach was successful in reducing T cell contamination of the graft and preventing acute and chronic GVHD. In this group of patients two suffered irreversible graft failure and one developed reversible graft failure. In a similarly sized group of patients previously transplanted with unpurged marrow according to the Seattle protocol, no episodes of graft failure occurred. Since other causes of graft failure, such as drug toxicity or viral infections, could be largely excluded, this suggested that the graft failures were specifically related to the purging process. In haploidentical bone marrow transplantation (BMT) O'Reilly has identified residual host-versus-graft activity (HVG) as a cause of graft failure. The causes and mechanisms of graft failure in T-depleted HLA-identical sibling transplants have not been extensively investigated to date. In the three graft failures observed by us, the loss of the graft was preceded by the appearance of a population of activated lymphocytes. We have determined the phenotype and origin of this population and investigated its interactions with donor hemopoietic tissue in vitro.     

Estimation of renal tubular secretion in man, in health and disease, using endogenous N-1-methylnicotinamide.

We have been investigating the possibility of using the renal clearance of endogenous N-1-methylnicotinamide (NMN) as a marker of renal tubular function. Sixty-three subjects (11 healthy volunteers and 52 patients with renal impairment) were used for this study. The subjects were divided into three groups according to their creatinine clearance: group 1, over 80 ml/min; group 2, between 30 and 80 ml/min, and group 3, less than 30 ml/min. The correlation between NMN and creatinine clearance was compared for each group. A good correlation (r = 0.84) was found for groups 1 and 3 (r = 0.76), whereas for group 2, a poor correlation was found (r = 0.43). For subjects with mild renal impairment (group 2), there was clear evidence of glomerulo-tubular imbalance manifest by a larger variability in NMN clearance than creatinine clearance and an essentially non-parallel decline in these two parameters for this group. When all subjects were grouped together, the relationship between the clearance of NMN and of creatinine was best described by a 3-term polynomial equation (r = 0.93). NMN clearance is a potentially useful non-invasive marker of renal tubular function in man and provides additional information to that provided by the measurement of creatinine clearance alone. This substance should be more fully evaluated as a potential diagnostic aid.