Phosphoenolpyruvate metabolism in Teladorsagia circumcincta: a critical junction between aerobic and anaerobic metabolism.

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L(3)) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L(3)s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L(3) and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.

The kinetics and regulation of phosphofructokinase from Teladorsagia circumcincta.

Phosphofructokinase (PFK-1) activity was examined in L(3) and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4±0.2 to 1.7±0.2 in L(3)s and 2.0±0.3 to 2.4±0.4 in adults) and the K(½) for fructose 6 phosphate (from 0.35±0.02 to 0.75±0.05mM in L(3)s and 0.40±0.03 to 0.65±0.05mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.

The tricarboxylic acid cycle in L3 Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA.

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L3 Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD+ and NADP+ specific) and α-ketoglutarate dehydrogenase in homogenates of L3 T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD+ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD+] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists withinL3 T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle inL3 T. circumcincta are probably similar to those in the tissues of their host species.

Teladorsagia circumcincta: survival of adults in vitro is enhanced by the presence of a mammalian cell line.

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO(2), 5% O(2) 85% N(2), 65% humidity at 37 degrees C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO(2) or O(2), or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.

Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta.

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30h and adult worms for 4-6h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30h. An initial external ammonia concentration of 600 microM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.

Progesterone stimulation of fluid absorption by the rat uterine gland.

The purpose of this study was to test the hypothesis that the steroid environment affects fluid absorption by the uterine glands. Laser scanning confocal microscopy of the distribution of an extracellular marker (fluorescein isothiocyanate-labelled dextran) within rat uterine glands showed that the endometrial glands change their fluid handling characteristics under different hormonal conditions. Under progesterone dominance, the glands showed an amiloride-sensitive dextran accumulation indicating sodium-dependent fluid absorption; however, this was absent in the oestrogen-dominated state. The rate of fluid uptake in the progesterone-stimulated gland opening was estimated to be approximately 1 x 10(-4) cm s(-1), requiring a suction pressure of between 10 and 20 mm Hg at the mucosal surface. This study provides the first direct evidence of fluid absorption by the uterine glands. Such absorption may provide the mechanism for closure of the uterine lumen and immobilization of the blastocyst necessary for implantation.

Evidence of amiloride-sensitive fluid absorption in rat descending colonic crypts from fluorescence recovery of FITC-labelled dextran after photobleaching.

1. Fluorescence recovery after photobleaching (FRAP) of fluorescein isothiocyanate (FITC)-labelled 10 and 250 kDa dextran (FITC dextran) in isolated rat descending colonic crypts was measured at 35 degrees C using laser scanning confocal microscopy. 2. FRAP of either 10 or 250 kDa FITC dextran in crypt lumens was almost complete within 2-3 min. 3. In the presence of amiloride (0.1 mM), or in the absence of Na+, the rate of FITC dextran uptake into the crypt lumens was reduced by 70-80 %. 4. The rate of fluid uptake into the crypt lumen, as estimated from the rate of total FITC dextran uptake into the crypt lumen and its adjacent pericryptal region after FRAP, was between 1.3 x 10(-3) and 1.7 x 10(-3) cm x s(-1). 5. Convective flow during FRAP was also determined from the initial rate of FITC dextran advance along the crypt lumen. This effect was almost completely blocked by amiloride (0.1 mM). 6. The permeability of 10 kDa FITC dextran across the descending colonic crypt wall was found to be higher than that of 250 kDa FITC dextran (3.7 (+/- 0.6) x 10(-5) and 1.8 (+/- 0.3) x 10(-6) cm x s(-1), respectively; n = 3 for both, P < 0.01). The permeability of the caecal crypt wall to 10 kDa dextran was higher than that of the descending crypt wall (2.03 (+/- 0.21) x 10(-5) cm x s(-1); n = 3, P < 0.025). 7. Simulation of the flow of Na+, water and FITC dextran into the crypt lumen and across the crypt wall and pericryptal sheath corroborates the observed parameters of water and Na+ flows.

Mechanisms of glucose transport at the blood-brain barrier: an in vitro study.

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood-brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood-brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood-brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood-brain barrier model of approximately 0.85. Under conditions where [glucose](lumen) was higher than [glucose](ablumen), influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.

Radiation induced cytochrome c release causes loss of rat colonic fluid absorption by damage to crypts and pericryptal myofibroblasts.

BACKGROUND: Therapeutic or accidental exposure to radiation commonly causes gastrointestinal disturbances, including diarrhoea. Rats subjected to whole body ionising radiation at a dose of 8 Gy lose their capacity to absorb fluid via the descending colon after four days. After seven days, fluid absorption recovers to control levels. AIMS: To investigate the effect of ionising radiation on colonic permeability together with its effect on mitochondria dependent apoptotic signals and intercellular adhesion molecules. METHODS: Rats were irradiated with doses of 0-12 Gy. Colonic permeability was measured by accumulation of fluorescein isothiocyanate (FITC) dextran in crypt lumens. Changes in levels of cytochrome c, caspase 3, E and OB cadherin, beta-catenin smooth muscle actin, and collagen IV were assessed using immunocytochemistry with confocal microscopy. RESULTS: Cytosolic cytochrome c increased after 8 Gy (t(1/2) 1.4 (0.6) hours) and peaked at approximately six hours. Caspase 3 increased more slowly, particularly in crypt epithelial cells (t(1/2) 57 (14.5) hours). Pericryptal myofibroblasts disintegrated within 24 hours as was evident from loss of OB cadherin and smooth muscle actin. This coincided with increased crypt permeability to dextran. Intercellular adhesion between crypt luminal cells was not lost until day 4 when both beta-catenin and E-cadherin were minimal. The half maximal dose-response for these effects was in the range 2-4 Gy. Recovery of colonic transport was concurrent with recovery of pericryptal smooth muscle actin and OB cadherin. The pan caspase inhibitor Z-Val-Ala-Asp.fluoromethylketone (1 mg/kg per day) had a small effect in conserving the pericryptal sheath myofibroblasts and sheath permeability but had no systemic therapeutic effects. CONCLUSIONS: These data suggest that radiation damage to the colon may be initiated by mitochondrial events. Loss of crypt fluid absorption and increased permeability coincided with decreased intercellular adhesion between crypt epithelial cells and loss of pericryptal sheath barrier function.

Vitamin C protects human vascular smooth muscle cells against apoptosis induced by moderately oxidized LDL containing high levels of lipid hydroperoxides.

Vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque "necrotic" core, cap rupture, and thrombosis. Oxidatively modified low-density lipoproteins (LDLs) are implicated in the pathogenesis of atherosclerosis, and dietary antioxidants are thought to protect the vasculature against LDL-induced cytotoxicity. Because LDL oxidative modification may vary within atherosclerotic lesions, we examined the effects of defined, oxidatively modified LDL species on human arterial smooth muscle cell apoptosis and the cytoprotective effects of vitamin C. Moderately oxidized LDL (0 to 300 microg protein/mL), which has the highest content of lipid hydroperoxides, induced smooth muscle cell apoptosis within 6 hours, whereas native LDL and mildly and highly oxidized LDL had no effect. Moderately oxidized LDL increased cellular DNA fragmentation, release of fragmented DNA into the culture medium, and annexin V binding and decreased mitochondrial dehydrogenase activity and expression of the antiapoptotic mediator Bcl-x(L). Treatment of cells with native LDL together with the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (HPODE, 200 micromol/L, 6 to 24 hours) also induced apoptotic cell death. Pretreatment of smooth muscle cells with vitamin C (0 to 100 micromol/L, 24 hours) attenuated the cytotoxicity and apoptosis induced by both moderately oxidized LDL and HPODE. Our findings suggest that moderately oxidized LDL, with its high lipid hydroperoxide content, rather than mildly or highly oxidized LDL, causes apoptosis of human smooth muscle cells and that vitamin C supplementation may provide protection against plaque instability in advanced atherosclerosis.

Regional differences in rat large intestinal crypt function in relation to dehydrating capacity in vivo.

1. Rat descending colon absorbed fluid against a large hydraulic resistance, imposed by 10 % agarose (w/v) gel plugs inserted in the lumen, by raising the tonicity of the absorbate from the gel to 880 +/- 54 mosmol kg-1; the tonicity of the absorbate from 2.5 % gels was 352 +/- 38 mosmol kg-1. The hypertonic absorbate generated an osmotic pressure which created a fluid tension in the crypt lumen. This was monitored as a suction tension in colonic luminal gels of 45.3 +/- 3 cmH2O with 2.5 % gels and 725 +/- 145 cmH2O with 10 % gels. The caecum was unable to absorb fluid against a significant hydraulic resistance. 2. Fluorescein isothiocyanate-labelled dextran (FITC dextran; molecular mass 10000 Da) accumulated within descending colonic crypt lumens by concentration polarization. Maximal accumulation at a depth of 20-40 micrometer below the mucosal surface was 5.68 +/- 0.2-fold above control levels. Caecal crypts accumulated dextran to a maximum of 1.8 +/- 0.17-fold above control levels. 3. The relationship between crypt luminal tension and suction tension of the distal colon was also demonstrated using paraffin, which occluded the crypt lumens with microscopic droplets and completely inhibited fluid absorption from high resistance luminal gels. 4. Reduction in dietary Na+ intake raised plasma aldosterone and the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. The caecum did not respond in this way to varied Na+ intake.

Regional crypt function in rat large intestine in relation to fluid absorption and growth of the pericryptal sheath.

1. Confocal microscopic studies of rat colonic mucosa showed that the pericryptal sheath surrounding distal colonic crypts is an effective barrier both to dextran and NaCl movement, whereas no such structure surrounds the caecal crypts. 2. The distal colonic pericryptal barrier was functionally demonstrated by accumulation of Sodium Green within the pericryptal space. After exposure to benzamil, Sodium Green accumulation was decreased. Fluorescein isocyanate-labelled dextran (FITC dextran; molecular mass 10000 Da) was accumulated in the crypt lumens and pericryptal spaces. Both dextran and Sodium Green accumulation were absent from the pericryptal zone surrounding caecal crypts. 3. Low dietary Na+ intake raised rat plasma aldosterone and stimulated distal pericryptal sheath growth and adhesiveness as shown by increased amounts of F-actin, smooth muscle actin, beta-catenin and E-cadherins in the pericryptal zone. It also raised the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. This linkage indicates that trophic effects on the colon resulting from a low Na+ diet are not confined solely to effects on transepithelial Na+ transport, but are observed in the pericryptal sheath. 4. A computer model of crypt function confirms that a pericryptal sheath with low permeability to NaCl is an essential component of the crypt dehydrating mechanism.

Applications of confocal and fluorescence microscopy.

Fluorescence microscopy has become a powerful tool for both the localization of cellular components in fixed cells, using target-specific fluorescent probes and labeled antibodies, and the fluorescence imaging of ions in single living cells. Despite its markedly lower spatial resolution when compared with electron microscopy, the essentially non-invasive nature of light microscopy provides a unique tool for examining cell behaviour at the level of the single cell bringing new insight into both cellular heterogeneities and cell-cell interactions. Further developments in both fluorescent probes and instrumentation should provide even more powerful tools to probe the mechanisms of cell function, both in vitro and in vivo.

Concentration polarization of fluorescent dyes in rat descending colonic crypts: evidence of crypt fluid absorption.

1. Using confocal microscopy, the rate of fluid absorption into isolated perifused descending rat colonic crypt lumens is estimated from the concentration polarization and distribution of fluorescein sulphonate (FS) and fluorescein isothiocyanate-dextran (FITC-dextran; molecular weight, 10,000) within the crypt lumens and pericryptal fluid. 2. The probe dyes enter the crypt via the luminal opening, are concentrated in the lumen, then escape into the pericryptal space via the paracellular spaces spanning the crypt wall. 3. FITC-dextran is maximally accumulated at a luminal depth of 60 microns to 5 times the concentration at the crypt opening (p < 0.001) and penetrates 150-200 microns along the lumen. FS is maximally accumulated within crypt lumen close to the opening. At crypt luminal depths 10-60 microns from the opening FS is accumulated by a factor of 1.5-2.0 above that found in HgCl2-treated tissue (p < 0.001). 4. Dye enters the crypt lumen slowly from the basal side, but from this side does not accumulate above the bathing solution concentration. 5. HgCl2 (20 microM) or theophylline (10 mM) completely inhibit concentrative accumulation of FITC-dextran and FS within the crypts and pericryptal space (p < 0.001). 6. Computer simulation of the dye uptake indicates that the rate of water flow into the crypt luminal opening is 1 x 10(-3) cm s-1 which is equivalent to 15 microliters (cm mucosa)-2 h-1. Approximately 75% of the fluid entering the crypt is abosrbed across the proximal 50 microns of crypt wall as a consequence of the large osmotic pressure gradient between the pericryptal and crypt luminal solutions. A pericryptal diffusion barrier with lower permeability than that across the crypt wall is required to simulate dye accumulation in the pericryptal space. Differences between FITC-dextran and FS accumulation are explained by the lower diffusion coefficient within the crypt lumen, and lower crypt wall permeability of FITC-dextran.

Direct observation of hexokinase translocation in stimulated macrophages.

1. Fluorescence imaging of antibodies was used to show that phorbol 12-myristate 13-acetate (PMA) induces a 4-fold increase in the amount of hexokinase relative to the control in the cortical shell of rat peritoneal macrophage cytosol adjacent to the plasma membrane, and a corresponding depletion in the amount of hexokinase in the central core of the cytosol. However, there was no significant PMA-dependent change in the distribution of glucose-6-phosphate dehydrogenase. 2. Cytochalasin D, an inhibitor of actin microfilament polymerization, prevented the PMA-induced hexokinase translocation and also reduced the PMA-dependent increases in 2-deoxy-D-glucose transport and glucose-dependent PMA-stimulated superoxide production. 3. PMA caused a contraction of the width of the cortical F-actin zone. Cytochalasin D caused some dispersal of F-actin within the cell, increasing the density of F-actin within the central cytosolic core and causing aggregation of the F-actin within the cortex. These data are consistent with the view that PMA induces attachment of hexokinase to microfilaments within the cortical zone adjacent to the cell membrane of macrophages, and cytochalasin D prevents this attachment. This is the first direct demonstration of the translocation of hexokinase to the plasma membrane in activated cells, and supports the view that enhanced hexokinase activity in the cortical region of the cytosol is an important early component of the macrophage activation process.

Evidence from fluorescence microscopy and comparative studies that rat, ovine and bovine colonic crypts are absorptive.

1. To test whether colonic crypts are secretory or absorptive interstitial [Na+] in rat descending colonic mucosa is determined using video-enhanced imaging of the impermeant acid form of the fluorescent Na+ probe SBFI (Molecular Probes) and intracellular [Na+] is monitored with SBFI (AM form). In rat descending colonic mucosa perifused with isotonic Tyrode solution interstitial [Na+] = 500-650 mM. Following exposure to Tyrode solution containing theophylline (10 mM) interstitial [Na+] falls by 300-450 mM within 1 min. Exposure to amiloride (0.2 mM) reduces the intracellular [Na+] from ca 25 to 12 mM within 15 min and concurrently decreases [Na+] in the interstitial fluid surrounding the crypts at the mucosal surface by approximately 200 mM. 2. The route of fluid inflow across the rat colonic mucosa is directly traced by perifusing with Tyrode solution containing the impermeant fluorescent dye, fluorescein disulphonate (FS). FS accumulates rapidly within crypt lumens of control tissues to a 2-fold higher concentration than in the external bathing solution, but FS does not accumulate in crypts of tissues treated with azide (2 mM). The increment in FS accumulation within the crypt lumen above the bulk solution decreases by 80% within 1 min following exposure to theophylline (10 mM), indicating that fluid absorption into crypts is reduced. Estimates of the total fluid influx from the rate and extent of FS concentration polarization within crypts indicate that it is sufficient to account for the entire transcolonic fluid absorption. 3. Comparative studies of isolated bovine and ovine colon were also undertaken to investigate the failure of bovine colon to generate a hypertonic absorbate and hence its incapacity to produce hard faeces. The interstitial fluid surrounding ovine colonic crypts is hypertonic to the bulk solution, whereas the interstitial fluid surrounding bovine colonic crypts is nearly isotonic with the bathing solution. Additionally, fluorescein disulphonate accumulates within ovine colonic crypt lumens by concentration polarization, whereas no concentration of FS occurs within bovine colonic crypt lumens. This corroborates the view that a hypertonic interstitial fluid is absent from bovine colon mainly because of a high rate of transepithelial leakage of low molecular weight solutes via paracellular routes.

Video enhanced imaging of the fluorescent Na+ probe SBFI indicates that colonic crypts absorb fluid by generating a hypertonic interstitial fluid.

Extracellular accumulation of Na+ detected by video-enhanced microscopic imaging of the impermeant fluorescent probe SBFI confirms the view that colonic crypts produce a hypertonic ascorbate ca 1000 mOsm.kg-1, thereby generating a large osmotic pressure across the crypt wall. This creates a high fluid tension within the crypt lumen, sufficient to dehydrate faeces. When bathed in Tyrode the SBFI.Na+ fluorescence indicates a [Na+] ca 750 mM within the interstitial space of metabolizing rat descending colon. There is no evidence of interstitial Na+ accumulation in octanol (2 mM) or in rabbit colon incubated with 1.0 mM ouabain and no evidence of Na+ secretion via the crypt lumen during absorption.

Regulation of insulin secretion from islets of Langerhans rendered permeable by electric discharge.

1. High-voltage electric discharge has been used to increase the permeability of B-cells of isolated islets of Langerhans to facilitate studies of the effects of normally impermeable substances on insulin secretion. 2. The application of an intense electric field increased the [(14)C]sucrose space of the islets from 37.8+/-3.1% to 86.2+/-5.2% of their total volume as assessed by (3)H(2)O content. The cells remained permeable for at least 40min. 3. Ultrastructural studies showed no deleterious changes in the structure of the B-cells after discharge. 4. Insulin secretion from normal islets was unaffected by increasing the medium [Ca(2+)] from 10nm to 10mum. In the islets that had been rendered permeable by discharge, insulin secretion was significantly increased under these conditions, without any alteration in the release of lactate dehydrogenase, a cytoplasmic marker enzyme. 5. Studies of the dynamics of insulin release during perifusion showed that the response to increased (10mum) Ca(2+) concentration was rapid and sustained over a period of at least 13min. 6. Secretion responses to Ca(2+) in perifusion established that maximum release in permeabilized islets occurs at approx. 1mum-Ca(2+) and half-maximum release occurs at approx. 0.6mum-Ca(2+). 7. The study of the effect of agents that interfere with the microtubular microfilamentous system in B-cells using a perifusion system revealed that cytochalasin B caused a considerable increase, whereas vinblastine sulphate caused a significant inhibition, in insulin release in response to 1mum-Ca(2+). 8. This technique should facilitate the study of the role of normally impermeable ions and metabolic intermediates in the regulation of insulin secretion.

Effect of enkephalins and morphine on insulin secretion from isolated rat islets.

The direct effects of an enkephalin analogue, (D-Ala2/MePhe4/Met/(O)-o1) enkephalin (DAMME), on insulin release from isolated islets of Langerhans of the rat have been investigated. DAMME had a dose-dependent effect on insulin secretion: low concentrations (10(-10) to 10(-8) mol/l) were stimulatory while high concentrations (10(-5) mol/l) were inhibitory in the presence of 8 mmol/l glucose. Similar effects were found with met-enkephalin, and with the longer acting alanine substituted met-enkephalin. Morphine sulphate (5 X 10(-7) mol/l) also stimulated insulin release. The effects of enkephalin and morphine were blocked by the specific opiate antagonist naloxone hydrochloride (1.2 X 10(-6) mol/l). The insulin secretory response of perifused islets to enkephalins and morphine was rapid, corresponding to the first phase of glucose induced insulin release. These observations suggest that there may be opiate receptors in islets, and that opioid peptides could modulate insulin release.