Acetylcholine Synthesis by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino)cyclohexanol (AH5183) and Picrylsulfonic Acid

The present experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol) and/or picrylsulfonic acid (TNBS), two compounds known to have the ability to block the uptake of ACh by cholinergic synaptic vesicles in vitro. We confirmed that, in stimulated (5 Hz) perfused (30 min) ganglia, AH5183 depressed ACh release and ACh tissue content increased by 86 +/- 6% compared to contralateral ganglia used as controls. Preganglionic activity increased ACh release by a similar amount in the presence (19.9 +/- 1.0 pmol/min) or absence (20.5 +/- 2.4 pmol/min) of TNBS. The final tissue ACh content was also similar in the presence (1,668 +/- 166 pmol) or absence (1,680 +/- 56 pmol) of TNBS. However, the AH5183-induced increase of tissue ACh content (86 +/- 6%) was abolished completely when AH5183 was perfused with 1.5 mM TNBS (-3.0 +/- 1.0%). This inhibition of ACh synthesis, observed in TNBS-AH5183-perfused ganglia, was not dependent upon further inhibition of ACh release beyond that caused by AH5183 alone, because 14.0 +/- 1.9% of the transmitter store was released by preganglionic nerve stimulation in the presence of TNBS plus AH5183 and this was similar in the presence of AH5183 without TNBS (14.0 +/- 0.6%). Moreover, when ganglia were first treated with TNBS and then stimulated in the presence of AH5183, an increase of 64 +/- 6% of the ganglionic ACh content occurred, and this increase was not statistically different from the increase measured with AH5183 alone (86 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Release of non-neuronal acetylcholine from the isolated human placenta is affected by antidepressants

Non-neuronal acetylcholine (ACh) is released from the human placenta into the extracellular space via organic cation transporters (OCTs). The present experiments investigated whether ACh release from epithelial cells is affected by drugs which are substrates of OCTs. The antidepressant drugs amitriptyline and doxepine were tested as both substances are not approved for pregnant women but frequently used. Release of ACh was measured in 10 min intervals over a period of 100 min. Test substances were added from t=50 min of incubation onwards. The effect was calculated by comparing the ACh release of the last three samples (t=70-100 min; B2) with that immediately before the application of the test substances (t=20-50 min; B1). The baseline ACh release amounted to 2.07+/-0.17 nmol/10 min (n=29; villus). Under control conditions a B2/B1 ratio of 0.78+/-0.02 was obtained. The following B2/B1 ratios were found, when therapeutic drugs were added: 0.54+/-0.04 (n=7; P<0.05) in the presence of 10 microM amitriptyline; 0.44+/-0.04 (10; P<0.01) in the presence of 10 microM doxepin; 0.73+/-0.04 (13) in the presence of 10 microM metformin; 0.76+/-0.06 (7) in the presence of 10 microM minoxidil; 0.63+/-0.03 (10) in the presence of 1 microM theophylline. The results demonstrate that antidepressants reduce the release of non-neuronal ACh at least in the human placenta, most likely by intracellular substrate competition at the polyspecific organic cation transporters (OCTs) but only at concentrations roughly 30-fold above the therapeutic range. Theophylline may also interfere with the release of non-neuronal ACh.     

Acetylcholine Synthesis and Release by a Sympathetic Ganglion in the Presence of 2-(4-Phenylpiperidino) Cyclohexanol (AH5183)

These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basal forebrain acetylcholine release during REM sleep is significantly greater than during waking

Cholinergic neurons of the basal forebrain supply the neocortex with ACh and play a major role in regulating behavioral arousal and cortical electroencephalographic activation. Cortical ACh release is greatest during waking and rapid eye movement (REM) sleep and reduced during non-REM (NREM) sleep. Loss of basal forebrain cholinergic neurons contributes to sleep disruption and to the cognitive deficits of many neurological disorders. ACh release within the basal forebrain previously has not been quantified during sleep. This study used in vivo microdialysis to test the hypothesis that basal forebrain ACh release varies as a function of sleep and waking. Cats were trained to sleep in a head-stable position, and dialysis samples were collected during polygraphically defined states of waking, NREM sleep, and REM sleep. Results from 22 experiments in four animals demonstrated that means +/- SE ACh release (pmol/10 min) was greatest during REM sleep (0.77 +/- 0.07), intermediate during waking (0.58 +/- 0.03), and lowest during NREM sleep (0.34 +/- 0.01). The finding that, during REM sleep, basal forebrain ACh release is significantly elevated over waking levels suggests a differential role for basal forebrain ACh during REM sleep and waking.     

Vagal stimulation suppresses ischemia-induced myocardial interstitial norepinephrine release

Although electrical vagal stimulation exerts beneficial effects on the ischemic heart such as an antiarrhythmic effect, whether it modulates norepinephrine (NE) and acetylcholine (ACh) releases in the ischemic myocardium remains unknown. To clarify the neural modulation in the ischemic region during vagal stimulation, we examined ischemia-induced NE and ACh releases in anesthetized and vagotomized cats. In a control group (VX, n = 8), occlusion of the left anterior descending coronary artery increased myocardial interstitial NE level from 0.46+/-0.09 to 83.2+/-17.6 nM at 30-45 min of ischemia (mean+/-SE). Vagal stimulation at 5 Hz (VS, n = 8) decreased heart rate by approximately 80 beats/min during the ischemic period and suppressed the NE release to 24.4+/-10.6 nM (P < 0.05 from the VX group). Fixed-rate ventricular pacing (VSP, n=8) abolished this vagally mediated suppression of ischemia-induced NE release. The vagal stimulation augmented ischemia-induced ACh release at 0-15 min of ischemia (VX: 11.1+/-2.1 vs. VS: 20.7+/-3.9 nM, P < 0.05). In the VSP group, the ACh release was not augmented. In conclusion, vagal stimulation suppressed the ischemia-induced NE release and augmented the initial increase in the ACh level. These modulations of NE and ACh levels in the ischemic myocardium may contribute to the beneficial effects of vagal stimulation on the heart during acute myocardial ischemia.     

Calcium dependent release of acetylcholine and gamma-aminobutyric acid from the rabbit retina.

The concurrent release of endogenous ACh and GABA from the retina (in the presence of physostigmine) was measured using either an eye-cup preparation in rabbits anaesthetized with urethane or isolated rabbit retinas. There was a spontaneous resting release of ACh and GABA from the dark adapted retina of ca 5 and 160 pmol min-1 respectively. Stimulation of the initially dark adapted retina in vivo with flickering light (0.1-20 Hz) increased the release of ACh by up to 5 times the spontaneous resting release but did not cause a detectable increase in GABA release. The maximum light-evoked release of ACh was about 24 pmol min-1/retina and occurred at a frequency of 10 Hz. However, the maximum release of ACh per flash occurred at 0.1 Hz at which frequency the average ACh release per flash from one amacrine cell was ca 2.35 x 10(-18) mol. Exposure of the retina to the potent inhibitors of GABA uptake, SKF89976A and SKF100330A markedly reduced the resting release of ACh and abolished the light-evoked release of ACh but did not enable a light-evoked release of GABA to be detected. Bicuculline blocked the inhibitory actions of both SKF89976A and SKF100330A on ACh release but the combination of bicuculline and uptake inhibitor did not result in a light-evoked release of GABA. In contrast, KCl (20 mM) applied locally to the retina in vivo resulted in the release of both ACh and GABA (61 and 2.6-fold respectively). KCl (20 mM) also evoked large increases in ACh and GABA release from isolated rabbit retinas in room light (13.5 and 3.4-fold respectively). The K-evoked release of ACh and GABA from the rabbit retina both in vivo and in vitro was calcium dependent. These experiments are the first in which endogenous ACh and GABA release from the retina have been simultaneously measured and suggest that the release mechanisms for these transmitters are fundamentally similar.     

Increases in cholesterol 7-hydroperoxides in lipids of human skin by sunlight exposure.

Free and ester forms of cholesterol 7alpha- and 7beta-hydroperoxides (Ch 7-OOHs) in skin lipids of humans were separated and determined by high performance liquid chromatography with a chemiluminescence detector. We first demonstrated the presence of Ch 7-OOHs in lipids of human skin. The levels of Ch 7-OOHs found in skin lipids of healthy Japanese volunteers (n = 5) ranged from 2.78 to 25.2 pmol/cm2 skin, indicating large inter-individual differences. However, the intra-individual differences of Ch 7-OOHs levels in skin lipids between right and left arms were less than 25% (-16.4% to 24.0%). Inter-day differences of Ch 7-OOHs in 5 subjects at 1 week interval were also small (-36.7% to 47.7%). Additionally, we investigated effects of sunlight exposure on the levels of Ch 7-OOHs in skin lipids of healthy Japanese volunteers (n = 24). The levels of Ch 7-OOHs in skin lipids significantly increased from 10.0+/-6.7 to 38.9+/-38.0 pmol/cm2 skin by sunlight exposure (10-40 mJ/cm2/min) for 3 h. Therefore, natural sunlight exposure causes lipid peroxidation in skin lipids of humans. These results suggest that the level of Ch 7-OOHs is a good marker for lipid peroxidation in human skin.     

Nitric oxide modulates the cardiovascular effects elicited by acetylcholine in the NTS of awake rats

Microinjection of acetylcholine chloride (ACh) in the nucleus of the solitary tract (NTS) of awake rats caused a transient and dose-dependent hypotension and bradycardia. Because it is known that cardiovascular reflexes are affected by nitric oxide (NO) produced in the NTS, we investigated whether these ACh-induced responses depend on NO in the NTS. Responses to ACh (500 pmol in 100 nl) were strongly reduced by ipsilateral microinjection of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 nmol in 100 nl) in the NTS: mean arterial pressure (MAP) fell by 50 +/- 5 mmHg before L-NAME to 9 +/- 4 mmHg, 10 min after L-NAME, and HR fell by 100 +/- 26 bpm before L-NAME to 20 +/- 10 bpm, 10 min after L-NAME (both P < 0.05). Microinjection of the selective inhibitor of neuronal nitric oxide synthase (nNOS), 1-(2-trifluoromethylphenyl) imidazole (TRIM; 13.3 nmol in 100 nl), in the NTS also reduced responses to ACh: MAP fell from 42 +/- 3 mmHg before TRIM to 27 +/- 6 mmHg, 10 min after TRIM (P < 0.05). TRIM also tended to reduce ACh-induced bradycardia, but this effect was not statistically significant. ACh-induced hypotension and bradycardia returned to control levels 30-45 min after NOS inhibition. Control injections with D-NAME and saline did not affect resting values or the response to ACh. In conclusion, injection of ACh into the NTS of conscious rats induces hypotension and bradycardia, and these effects may be mediated at least partly by NO produced in NTS neurons.     

Effect of bile salts on the plasma concentration of immunoreactive vasoactive intestinal polypeptide in man

The effects of bile salts on the release of immunoreactive vasoactive intestinal polypeptide (IR-VIP) were investigated in men using a specific radioimmunoassay. Plasma IR-VIP was determined after extraction by the acid-acetone method (recovery 75 +/- 5%). Oral administration of 400 mg sodium taurocholate caused a rise in plasma IR-VIP from 18.5 +/- 1.3 pmol/l to 31.1 +/- 2.1 pmol/l after 30 min and 39.0 +/- 1.7 pmol/l after 60 min and return to the initial value after 120 min. Oral administration of chenodeoxycholic acid (CDCA) also increased plasma IR-VIP from a basal level of 14.5 +/- 1.5 pmol/l to 36.3 +/- 1.2 pmol/l after 60 min. Oral administration of ursodeoxycholic acid (UDCA) increased plasma IR-VIP from 11.9 +/- 1.1 pmol/l to 25.6 +/- 1.8 pmol/l after 30 min. Perifusion of 1 mM taurocholate stimulated release of IR-VIP from human duodenal mucosa into the perifusate. These results suggest that bile salts may participate, at least in part, in the release of IR-VIP from the gut.     

Acetylcholine release from the carotid body by hypoxia: evidence for the involvement of autoinhibitory receptors.

The purpose of the present study was to investigate whether hypoxia influences acetylcholine (ACh) release from the rabbit carotid body and, if so, to determine the mechanism(s) associated with this response. ACh is expressed in the rabbit carotid body (5.6 +/- 1.3 pmol/carotid body) as evidenced by electrochemical analysis. Immunocytochemical analysis of the primary cultures of the carotid body with antibody specific to ACh further showed that ACh-like immunoreactivity is localized to many glomus cells. The effect of hypoxia on ACh release was examined in ex vivo carotid bodies harvested from anesthetized rabbits. The basal release of ACh during normoxia ( approximately 150 Torr) averaged 5.9 +/- 0.5 fmol.min-1.carotid body-1. Lowering the Po2 to 90 and 20 Torr progressively decreased ACh release by approximately 15 and approximately 68%, respectively. ACh release returned to the basal value on reoxygenation. Simultaneous monitoring of dopamine showed a sixfold increase in dopamine release during hypoxia. Hypercapnia (21% O2 + 10% CO2) as well as high K+ (100 mM) facilitated ACh release from the carotid body, suggesting that hypoxia-induced inhibition of ACh release is not due to deterioration of the carotid body. Hypoxia had no significant effect on acetylcholinesterase activity in the medium, implying that increased hydrolysis of ACh does not account for hypoxia-induced inhibition of ACh release. In the presence of either atropine (10 microM) or domperidone (10 microM), hypoxia stimulated ACh release. These results demonstrate that glomus cells of the rabbit carotid body express ACh and that hypoxia overall inhibits ACh release via activation of muscarinic and dopaminergic autoinhibitory receptors in the carotid body.     

Acetylcholine release and choline uptake by cuttlefish (Sepia officinalis) optic lobe synaptosomes

Acetylcholine (ACh), which is synthesized from choline (Ch), is believed to hold a central place in signaling mechanisms within the central nervous system (CNS) of cuttlefish (Sepia officinalis) and other coleoid cephalopods. Although the main elements required for cholinergic function have been identified in cephalopods, the transmembrane translocation events promoting the release of ACh and the uptake of Ch remain largely unsolved. The ACh release and Ch uptake were quantitatively studied through the use of in vitro chemiluminescence and isotopic methods on a subcellular fraction enriched in synaptic nerve endings (synaptosomes) isolated from cuttlefish optic lobe. The ACh release evoked by K+ depolarization was found to be very high (0.04 pmol ACh.s(-1).mg(-1) protein). In response to stimulation by veratridine, a secretagogue (a substance that induces secretion) that targets voltage-gated Na+ channels, the release rate and the total amount of ACh released were significantly lower, by 10-fold, than the response induced by KCl. The high-affinity uptake of choline was also very high (31 pmol Ch.min(-1).mg(-1) protein). The observed ACh release and Ch uptake patterns are in good agreement with published data on preparations characterized by high levels of ACh metabolism, adding further evidence that ACh acts as a neurotransmitter in cuttlefish optic lobe.     

In vivo and in vitro evidence for ACh-stimulated L-arginine uptake

Whereas L-arginine is clearly recognized as the precursor for nitric oxide synthesis, and its entry into endothelial cells via system y(+) transport is established, few data exist regarding the acute regulation of this transport process. We specifically investigated the effect of ACh and isoprenaline (Iso) on L-arginine uptake in the human forearm and in cultured bovine aortic endothelial cells (BAEC). Sixteen healthy males were studied. During a steady-state intra-arterial infusion of [(3)H]L-arginine (100 nCi/min), the effects of ACh (9.25 and 37 microg/min), Iso (25-50 and 200 microg/min), and sodium nitroprusside (SNP) (1-2 and 8 microg/min) on forearm plasma flow (FPF), L-[(3)H]arginine uptake, and L-[(3)H]citrulline release were determined. In parallel experiments, the effects of ACh, Iso, and SNP on L-[(3)H]arginine uptake were studied in BAEC. L-Arginine uptake was inversely related to FPF (r = -0.50; P < 0.005). At a similar FPF (ACh 56.82 +/- 9.25, Iso 58.49 +/- 5.56, SNP 57.92 +/- 4.96 ml/min; P = ns), intra-arterial ACh significantly increased forearm uptake of L-[(3)H]arginine (54,655 +/- 8,018 dpm/min), compared with that observed with either Iso (40,517.23 +/- 6,841 dpm/min; P = 0.01) or SNP (36,816 +/- 4,650 dpm/min; P = 0.011). This was associated with increased ACh-induced L-[(3)H]citrulline release compared with Iso and SNP (P = 0.046). Similarly, in BAEC, ACh significantly increased L-[(3)H]arginine uptake compared with control, Iso, or SNP (ACh 12.0 x 10(7) +/- 1.83 x 10(7) vs. control 6.67 x 10(7) +/- 1.16 x 10(7) vs. Iso 7.35 x 10(7) +/- 1.63 x 10(7) vs. SNP 6.01 x 10(7) +/- 1.11 x 10(7) fmol.min(-1).mg(-1) at 300 micromol/l L-arginine; P = 0.043). Taken together, these data indicate that ACh stimulates L-arginine uptake in cultured endothelial cells and in human forearm circulation, indicating the potential for acute modulation of endothelial L-arginine uptake.     

Dysfunctional inhibitory muscarinic receptors mediate enhanced histamine release in isolated human bronchi

In human airways mucosal mast cells are under the control of inhibitory muscarinic receptors. The described experiments tested, whether the inhibitory potency of two muscarinic receptor agonists (oxotremorine, acetylcholine) becomes impaired in advanced chronic obstructive pulmonary disease (COPD). Isolated human bronchi obtained from 26 patients with lung cancer were separated into two groups. Group 1 patients suffered from moderate COPD (mean FEV1 56%; range 34-71%; mean pack years of cigarette smoking 50, range 20-96; one non-smoker). Group 2 patients had no or only a mild form of COPD; mean FEV1 was 82% (62-97%) and the number of pack years 22 (6-45; 3 non-smoker). The calcium ionophore A23187 induced a maximal histamine release of 4100+/-870 pmol/g/5 min in group 1 bronchi, in contrast to only 1730+/-240 pmol/g/5 min in group 2 bronchi (p<0.02). Oxotremorine (1 nmol/L) reduced the stimulated histamine release by 81+/-5% in group 2 bronchi, but did not produce a significant effect in group 1 bronchi (11+/-14%). In conclusion, the present experiments show an enhanced histamine release in advanced COPD, which can be explained by a dysfunction of inhibitory muscarinic receptors.     

Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity

Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.     

Synergistic effect of angiotensin-(1-7) on bradykinin arteriolar dilation in vivo

The interaction between angiotensin [Ang-(1-7)] and bradykinin (BK) was determined in the mesentery of anesthetized Wistar rats using intravital microscopy. Topical application of BK and Ang-(1-7) induced vasodilation that was abolished by the BK B2 receptor antagonist HOE-140 and the Ang-(1-7) antagonist A-779, respectively. BK (1 pmol)-induced vasodilation, but not SNP and ACh responses, was potentiated by Ang-(1-7) 10 pmol and 100 pmols. The effect of 100 pmol of Ang-(1-7) on BK-induced vasodilation was abolished by A-779, indomethacin, and L-nitroarginine methyl esther, whereas losartan was without effect. Enalaprilat treatment enhanced the BK- and Ang-(1-7)-induced vasodilation and the potentiating effect of Ang-(1-7) on BK vasodilation. The potentiation of BK-induced vasodilation by Ang-(1-7) is a receptor-mediated phenomenon dependent on cyclooxygenase-related products and NO release.     

Glucose dependent insulinotropic polypeptide (GIP) infused intravenously is insulinotropic in the fasting state in type 2 (non-insulin dependent) diabetes mellitus

The effects of an intravenous infusion of porcine GIP on beta-cell secretion in patients with untreated type 2 diabetes mellitus have been studied. The subjects were studied on two separate days. After a 10 h overnight fast and a further 120 min basal period they were given an intravenous infusion of porcine GIP (2 pmol.kg-1.min-1) or control solution in random order from 120-140 min. Frequent plasma glucose, insulin, C-peptide and GIP measurements were made throughout and the study was continued until 200 min. Plasma glucose levels were similar throughout both tests. During the GIP infusion there was an early significant rise in insulin concentration from 0.058 +/- 0.006 nmol/l to 0.106 +/- 0.007 nmol/l (P less than 0.01) within 6 min of commencing the GIP infusion and insulin levels reached a peak of 0.131 +/- 0.011 nmol/l at 10 min (P less than 0.01). Insulin levels remained significantly elevated during the rest of the GIP infusion (P less than 0.01-0.001) and returned to basal values 20 min post infusion. No change in basal insulin values was seen during the control infusion. C-peptide levels were similarly raised during the GIP infusion and the increase was significant just 4 min after commencing the GIP infusion (P less than 0.05). GIP levels increased from 16 +/- 3 pmol/l prior to the infusion to a peak of 286 +/- 24 pmol/l 20 min later. At 4 min when a significant beta-cell response was observed GIP levels were well within the physiological range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Persistent decreased plasma cholecystokinin levels in celiac patients under gluten-free diet: respective roles of histological changes and nutrient hydrolysis

Celiac disease is associated with impaired cholecystokinin (CCK) release. The mechanism by which CCK release is impaired is poorly understood and seems to be related to the mucosal atrophy or to decreased stimulation due to reduced intraduodenal nutrient hydrolysis. The aims of our study were to evaluate basal and postprandial CCK in celiac patients presenting with distinctive types of mucosal lesions (normal, infiltrative and atrophic), and to study the role of protein hydrolysis on CCK release. Plasma CCK was measured in 20 celiac patients (normal mucosa: n=6; infiltrative type: n=6; atrophic type=8) and 9 controls, before and after ingestion of a polymeric or a semi-elemental meal. Significant decreases in basal CCK plasma (B 0.6 [95% CI, 0.3-1.3] pmol/l; p<0.003) and postprandial CCK area under curve (AUC 34 [19-61] pmol/l x 120 min, p<0.0001) were observed in patients with an atrophic mucosa compared with treated patients (B 1.6 [1.0-2.4] pmol/l, AUC 267 [172-414] pmol/l x 120 min) or healthy volunteers (B 1.0 [0.7-1.4] pmol/l, AUC 186 [131-264] pmol/l x 120 min). A significant defective CCK release was also observed in patients with an infiltrative type: B 0.4 [0.2-0.7] pmol/l and AUC 56 [31-101] pmol/l x 120 min; p<0.0001. Administration of a semi-elemental diet did not correct the defective CCK release. In conclusion, the decreased CCK levels observed in celiac patients are not strictly related to the mucosal atrophy but rather to the lymphocytic infiltrate. Administration of a predigested meal did not correct the impaired CCK release. Some inhibitory mechanism could be involved in the CCK cell dysfunction observed in celiac patients presenting with lesser degrees of disease activity.     

Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors

Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (K(m)=200+/-20 nM, V(max)=25+/-3 pmol min(-1) mg protein(-1)), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (K(m)=5.0+/-0.5 microM, V(max)=160+/-15 pmol min(-1) mg protein(-1)) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells.     

Fragments of functional proteins in a primary culture of human erythrocytes

According to the previously reported data, the superntant of the primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic β-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, and amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age and blood group of donors. The dynamics of accumulation in the supernatant (20–80 min of incubation) of 14 hemoglobin fragments with the most reliably reproducible contents was obtained. The content of six peptides increased more than twofold between 20 and 40 min of incubation; the maximum increase in concentration was observed between 40 and 80 min (140%). The level of peptides that had the maximum concentration at the end of incubation was about 1000 pmol/ml of sedimented erythrocytes. The biological effects of the peptides identified in the supernatant of erythrocytes involve the stimulation of proliferation and hemopoiesis, suppression of proliferation, a bactericide effect, etc. These effects indicate the physiological importance of the peptide release by erythrocytes.     

Fragments of functional proteins in the primary culture of human erythrocytes

According to previously reported data, the supernatant of a primary culture of human erythrocytes contains 33 hemoglobin fragments. An analysis of the supernatant of a 20% (v/v) suspension of human erythrocytes allowed us to identify additionally four peptides whose precursors are cytoplasmic beta-actin (two fragments), fructose diphosphate aldolase B, and an unknown protein, as well as the amino acids tyrosine and tryptophan. The composition and the content of the components of the supernatant did not depend on the age or blood group of donors. The dynamics of accumulation in the supernatant (20-80 min of incubation) of the 14 hemoglobin fragments with the most reliably reproducible contents was obtained. The content of six peptides increased more than twofold between 20 and 40 min of incubation: the maximum increase in concentration was observed between 40 and 80 min (140%). The level of peptides that had the maximum concentration at the end of incubation was about 1000 pmol/ml of sedimented erythrocytes. The biological effects of the peptides identified in the supernatant of erythrocytes involve the stimulation of proliferation and hemopoiesis, suppression of proliferation, a bactericide effect, etc. These effects indicate the physiological importance of peptide release by erythrocytes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.