Release of [3H]acetylcholine from a modified rat phrenic nerve-hemidiaphragm preparation

Summary Two different preparations of the rat phrenic nerve-hemidiaphragm (whole nerve-muscle preparation, end-plate preparation) were used for studying synthesis and release of radioactive acetylcholine in the absence and presence of cholinesterase inhibitors.When the whole nerve-muscle preparation (110–180 mg) was incubated with [³H]choline, only small amounts of radioactive acetylcholine were synthesized within the tissue. Electrical nerve stimulation of the whole nerve-muscle preparation produced no increase in tritium outflow.Incubation of the end-plate preparation (16–29 mg) which was obtained after removal of most of the muscle mass led to the formation of large amounts of [³H]acetylcholine. Synthesis depended on nerve activity and increased 13-fold during a high loading stimulation (50 Hz), as compared to the synthesis at rest. In a denervated end-plate preparation the formation of [³H]acetylcholine was reduced to 4% of the control preparation. Electrical nerve stimulation of the end-plate preparation produced a release of tritium that could be attributed entirely to the release of [³H]acetylcholine. The stimulated tritium efflux was completely suppressed in a calcium-free medium or in the presence of tetrodotoxin (300 nM). Release could even be detected during a short train of 50 pulses (5 Hz) with a fractional release of about 0.04% of the [³H]acetylcholine tissue content per pulse.It is concluded that the large muscle mass interferes with nerve labelling by a reduction of the [³H]choline supply to the nerve terminals when the whole nerve-muscle preparation is used. Removal of most of the muscle fibres reduces the possibility for [³H]choline to be captured by them and then more radioactive choline can enter the end-plate region. From this end-plate preparation a calcium-dependent release of radioactive transmitter can be measured in the absence of cholinesterase inhibitors.     

Uptake and metabolism of [3H]choline by the rat phrenic nerve-hemidiaphragm preparation

Summary A whole nerve-muscle preparation (about 160 mg) or an end-plate preparation (about 25 mg) of the rat phrenic nerve-hemidiaphragm were incubated with [³H]choline, to investigate choline uptake and choline metabolism. Choline uptake was measured from the disappearance of choline from the incubation medium during the loading period and from the retention of tritium in the tissue after the loading and washout period.Based on the results obtained with both methods the end-plate preparation takes up three times as much choline than the whole nerve-muscle preparation or a small muscle strip that was cut outside the end-plate region and had a similar size as the end-plate preparation. Choline uptake was not markedly affected by the degree of nerve activity or by a chronic denervation. However, hemicholinium-3 significantly reduced (50%) the choline uptake by the end-plate preparation.Most of the choline (70–88%) taken up was metabolized and incorporated into membrane structures. Phosphatidylcholine was the predominant metabolite in both preparations. The ratio of phosphatidylcholine/lysophosphatidylcholine in the end-plate preparation (16) was significantly lower than in the whole nerve-muscle preparation (31). This might indicate a higher metabolism of phosphatidylcholine in the end-plate preparation.It is suggested that choline uptake by the rat phrenic nerve-hemidiaphragm occurs mainly by the muscle fibres. The innervated part of the muscle fibres can accumulate more choline than the peripheral part outside the end-plate region, probably because of a very active choline phospholipid metabolism within the end-plate region. Send offprint requests to I. Wessler at the above address     

First-pass metabolism of salicylamide. Studies in the once-through vascularly perfused rat intestine-liver preparation

Salicylamide (SAM) metabolism was studied in a once-through in situ perfused rat intestine-liver preparation in a manner which mimicked the first-pass effect. SAM (40 or 200 microM) was delivered into the intestine via the superior mesenteric artery at a flow rate of 7.5 ml/min. The intestine venous outflow into the portal vein and the hepatic arterial flow (2.5 ml/min; without drug) served as dual inflows into the liver. The steady state intestinal and hepatic extraction ratios were 0.262 +/- 0.055 and 0.992 +/- 0.014, respectively, at 40 microM, and 0.206 +/- 0.035 and 0.638 +/- 0.117, respectively, at 200 microM. SAM glucuronide was found to be the only metabolite formed by the intestine at both doses. Less than 3% of the dose was secreted into the intestinal lumen, with SAM glucuronide and SAM as the major and minor components, respectively. Hepatic metabolism of SAM, however, revealed SAM sulfation as the predominant pathway, while glucuronidation and hydroxylation were minor metabolic pathways. About 6% of the dose was excreted into bile, mostly as SAM and gentisamide glucuronides. The interrelationship between the intestine and liver clearances was also examined by mass balance considerations and simulation of data. The total rate of elimination of substrate across the two organs is the sum of the rates of metabolism by each organ. However, the overall effective extraction ratio and, hence, the clearance, are less than the sum of the individual extraction ratios and organ clearances, respectively. Our results showed that intestinal metabolism regulated the available substrate for hepatic elimination, and hence modified the contribution of hepatic metabolism in the overall first-pass effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction between divalent cations and botulinum toxin type A in the paralysis of the rat phrenic nerve-hemidiaphragm preparation

1. The effects of divalent cations (Ca²⁺, Sr²⁺ and Mg²⁺) on botulinum toxin paralysis of rat phrenic nerve-hemidiaphragm preparations have been investigated. Variations in divalent cation concentration were intended to modify transmitter release from nerve terminals.2. Onset of botulinum toxin activity was found to be dependent upon the process of transmitter release. Either Ca²⁺ or Sr²⁺ could initiate toxin activity. Tissues bathed in medium devoid of added Ca²⁺ or Sr²⁺ did not undergo botulinum paralysis. Likewise, preparations bathed in Ca²⁺ or Sr²⁺ plus high Mg²⁺ were resistant to toxin action.3. After onset of toxin activity, transmitter release acted to antagonize botulinum paralysis. Thus, high levels of Ca²⁺ or Sr²⁺ caused prolongation of paralysis times of toxin treated neuromuscular preparations.4. A partial model is proposed to explain the interaction between the mechanism of transmitter release and botulinum toxin paralysis.     

Release of substance P-like immunoreactivity from the vascularly perfused rat stomach

The release of gastric substance P-like immunoreactivity (SP-LI) has been studied in the vascularly perfused rat stomach. In the presence of 20 μM bacitracin and captopril, basal release of SP-LI was sustained throughout the experiments. Gastric SP-LI release was stimulated in a concentration-dependent manner by increasing the concentration of KCl in the perfusion medium. This stimulated release was reduced by the omission of Ca²⁺, indicating that a Ca²⁺-dependent mechanism was involved. Naloxone did not alter basal SP-LI secretion. [Met⁵]Enkephalin also had no significant effect on K⁺-stimulated secretion suggesting that enkephalinergic mechanisms are not involved. Gastric SP-LI release was also increased by capsaicin perfusion but this was not sustained. In conclusion, the present results provide the first evidence for the release of SP-LI into the rat stomach vasculature.     

Effects of sodium 5-methoxysalicylate on macromolecule absorption and mucosal morphology in a vascularly perfused rat gut preparation in vivo

The effect of sodium 5-methoxysalicylate on absorption was assessed by measurement of the appearance of test compounds in the portal blood output of a perfused rat gut model. The test compounds were a hexapeptide analogue of somatostatin, insulin, and horseradish peroxidase. Considerable amounts of sodium 5-methoxysalicylate were present in the portal blood 10 min after intraduodenal administration. When co-administered with sodium 5-methoxysalicylate (60 mg), a marked increase in the concentrations of the test substances occurred at t = 15 min which lasted for a further 15 min. Quantities of less than 60 mg had much reduced adjuvant effects. In control experiments with no adjuvant, the concentrations of the test substances remained low throughout the 1-h period of blood collection. After the administration of 60 mg of sodium 5-methoxysalicylate, slight mucosal damage was apparent at 10 min. This became progressively worse with time and, at 1 h, extensive mucosal stripping had occurred. The results suggest, although they do not prove, that apparent adjuvant effects in the small intestine may be a direct consequence of serious mucosal damage. This means that care must be taken in the investigation of adjuvant properties to exclude the possibility that an observed increase in transport is due to gross loss of integrity of the membrane.     

Absorption of benzoic acid in segmental regions of the vascularly perfused rat small intestine preparation.

Oral bioavailability is a consequence of intestinal absorption, exsorption, and metabolism and is further modulated by the difference in activities among segmental regions. The influence of these factors on the net absorption of benzoic acid (BA), a substrate that is metabolized to hippurate and is transported by the monocarboxylic acid transporter 1, was studied in the recirculating, vascularly perfused, rat small intestine preparation. Metabolism of BA was not observed for both systemic and intraluminal injections into segments of varying lengths. But, secretion of BA into lumen was noted. Absorption of BA (0.166-3.68 micromol) introduced at the duodenal end for absorption by the entire intestine was complete (>95% dose at 2 h) and dose-independent, yielding similar absorption rate constants (k(a) of 0.0464 min(-1)). The extent of absorption remained high (92-96% dose) when BA was injected into closed segments of shorter lengths (12 or 20 cm), suggesting a large reserve length of the rat intestine. However, k(a) was higher for the jejunum (0.0519 and 0.0564 min(-1), respectively, for the 12- and 20-cm segments) and exceeded that for the duodenum (12-cm segment, 0.0442 min(-1)) and ileum (20-cm segment, 0.0380 min(-1)) at closed injection sites. The finding paralleled the distribution of monocarboxylic acid transporter isoform 1 detected by Western blotting along the length of the small intestine. Fits of the systemic and oral data (based on duodenal injection for absorption by the whole intestine) to the traditional, physiological model and to the segregated flow model (SFM) that describes partial intestinal flow to the enterocyte region showed a better fit with the SFM even though metabolite data were absent.     

Succinylcholine-induced acceleration and suppression of electrically evoked acetylcholine release from mouse phrenic nerve-hemidiaphragm muscle preparation.

The effects of a depolarizing neuromuscular blocker on electrically evoked acetylcholine (ACh) release were studied using a mouse phrenic nerve-diaphragm muscle preparation preloaded with [3H]-choline, and the changes in muscle tension were recorded simultaneously. Succinylcholine at a low concentration (1 microM) enhanced evoked [3H]-ACh release, which tended to follow the increase in peak amplitude of tetanic tension; whereas at high concentrations (10 and 30 microM), it simultaneously reduced both release and tension. Decamethonium even at 10 and 30 microM had little effect on [3H]-ACh release despite producing a significantly greater reduction in tension compared with succinylcholine. (+)-Tubocurarine (5 microM) prevented the enhancing effect of [3H]-ACh release induced by 1 microM, but not the decreasing effect induced by 10 microM succinylcholine. These results suggest that succinylcholine induced acceleration at low concentrations due to a positive feedback mechanism through presynaptic nicotinic ACh receptors and the inhibition of ACh release at high concentration contributes in part to the neuromuscular blockade.     

Effects of atropine and glycopyrrolate on neuromuscular transmission in the rat phrenic nerve-diaphragm preparation

1. The effects of atropine and glycopyrrolate on neuromuscular transmission and on muscle contraction, were studied, in the rat diaphragm preparation, by analyzing their effects on the indirectly (and directly)-elicited twitch (0.2 Hz), tetanic (50 Hz for 20 sec duration), post-tetanic twitch responses (at 5 sec after the tetanus), and on the phenomenon of post-tetanic twitch potentiation (PTP), which is thought to be of a presynaptic origin, i.e. due to increased transmitter release. 2. Atropine (0.001-10 microM) increased the indirectly-elicited twitch tension by 22 +/- 2.1% (control 0.9 +/- 0.1 g, P less than 0.02), the tetanus by 15 +/- 1.1% (control 3.9 +/- 0.7 g, P less than 0.05), the post-tetanic twitch response by 33 +/- 3.1% (control 1.2 +/- 0.1 g, P less than 0.01) and the PTP value by 36 +/- 1.9% (control 33 +/- 2.3%, P less than 0.01, means +/- SEM = 6). 3. Atropine (0.001-10 microM) had little effect on the directly-elicited twitch tension, but in high concentrations (e.g. 20 microM), it blocked the twitch tension. 4. In contrast, glycopyrrolate (0.1-100 microM) had little effect on the twitch tension (direct or indirect), but it significantly reduced the tetanus (by 38 +/- 3.5%, P less than 0.01), the post-tetanic twitch response (by 17 +/- 1.2%, P less than 0.05) and the PTP values (by 24 +/- 3.1% P less than 0.02). 5. In the presence of hemicholinium (1.3 microM) the responses to atropine and glycopyrrolate were altered (decreased), indicating a possible action on presynaptic mechanism of transmission. 6. It is concluded that atropine and glycopyrrolate produce different (opposite) effects at the rat neuromuscular junction, atropine enhances whereas glycopyrrolate depresses neuromuscular transmission. The effects of these two antimuscarinic drugs may be exerted at the presynaptic nerve terminals, i.e. on presynaptic muscarinic receptors, which are involved in the feedback mechanism of transmitter release.     

A perfused tail artery preparation from the rat

The isolated perfused tail artery of the rat responds by constriction to 1·0 ng (-)-noradrenaline, and would be suitable for the assay of sympathomimetic amines. Electrical stimulation of this preparation is shown to activate solely postganglionic adrenergic nerve terminals. Tachyphyllaxis to angiotensin, vasopressin and bradykinin preclude the use of this preparation for their assay.     

First-pass metabolism of gentisamide: influence of intestinal metabolism on hepatic formation of conjugates. Studies in the once-through vascularly perfused rat intestine-liver preparation

Gentisamide (GAM) metabolism was studied in a once-through in situ perfused rat intestine-liver preparation in a manner which mimicked the first-pass effect. GAM (2.56 to 392 microM) was delivered into the intestine via the superior mesenteric artery at a flow of 7.5 ml/min. The intestinal venous outflow into the portal vein and the hepatic arterial flow (2.5 ml/min; without drug) served as dual inflows into the liver. The steady state intestinal extraction ratios (E1, 0.33 to 0.06) and hepatic extraction ratios (EH, 0.84 to 0.37) were highly concentration dependent, decreasing with increasing GAM concentrations. Gentisamide-5-glucuronide was found to be the major intestinal metabolite, and gentisamide-2-sulfate was also formed, albeit to a much lesser extent. However, hepatic metabolism of GAM revealed 5-sulfation and glucuronidation as the predominant pathways, with GAM 2-sulfation as a minor pathway. About 1 to 3% and 7% of dose was excreted into lumen and bile, respectively, mostly as GAM-5G. From the metabolic data, the fitted parameters confirmed that more enzymes exist in the liver than in intestine for the processing of GAM. The Km for 2-sulfation by the intestine was higher than the apparent Kms observed for 2- and 5-sulfation in liver. However, the Kms for 5-glucuronidation for both the intestine and liver were similar. The interrelationship between intestine and liver formation of first-pass metabolites showed that, at low GAM input concentration entering the IL preparation, the intestine, being the anterior organ, regulated the available substrate for hepatic elimination, and hence reduced the contribution of hepatic metabolism in the overall first-pass effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

基于大鼠原位肠-肝血管灌流模型研究甘草酸的代谢

目的研究甘草酸在大鼠体内的肠、肝中的生物转化。方法建立大鼠原位肠-肝血管灌流模型,采用LC-MS/MS方法测定灌流液中的甘草酸和甘草次酸。结果甘草酸在单向肠-肝血管灌流模型中稳态肠提取率和稳态肝提取率分别为(4.2±0.6)%和(28.0±3.0)%,灌流液中未发现代谢物甘草次酸;原位循环肠血管灌流模型中甘草酸的一级吸收速率常数为(0.33±0.06)min-1;甘草酸在大鼠十二指肠给药后,灌流液中主要活性代谢产物为甘草次酸,同时肠腔液也存在大量甘草次酸。结论甘草酸的首过效应明显,口服后其主要被肠道菌群或肝细胞代谢,在肠粘膜细胞仅少量代谢。大鼠原位肠-肝血管灌流模型适用于甘草酸的药动学研究。     

The release of prostaglandin E1 from the rat phrenic nerve-diaphragm preparation

1. The release of prostaglandin E(1) from the rat phrenic nerve-diaphragm preparation during tetanic contraction of the muscle in response to nerve stimulation is reported. Tentative identification of the prostaglandin depended on solvent extraction column and thin-layer chromatography and parallel biological assay.2. Polar lipid substances were released from the preparation in the absence of nerve stimulation by (+)-tubocurarine and noradrenaline.3. The output on nerve stimulation was not abolished by (+)-tubocurarine, hemicholinium, bretylium or phenoxybenzamine added to the bath.4. The possible origin of these prostaglandins is discussed.     

Histamine release in the isolated vascularly perfused stomach of the rat: regulation by autoreceptors.

1. In the isolated vascularly-perfused stomach of the rat, gastrin 1-17 (520 pmol 1(-1)) increased acid output from basal values of 13.7 +/- 2.7 to 92.5 +/- 11.4 mumol h-1 and venous histamine output from 10.1 +/- 2.3 to 54.7 +/- 7.9 nmol h-1 (mean +/- s.e.mean). 2. The H1 receptor agonist 2-methylhistamine (10 mumol 1(-1)) increased acid output to 21.6 +/- 2.9 mumol h-1 (P less than 0.05) and reduced basal histamine output to 4.0 +/- 0.8 nmol h-1 (P less than 0.05). Gastrin-stimulated acid secretion and vascular histamine output was not significantly affected by 2-methylhistamine (10 mumol 1(-1)). 3. The H2 receptor agonist, impromidine, dose-dependently increased basal acid secretion, reaching a maximal value of 145.5 +/- 11.7 mumol h-1 with impromidine (10 mumol 1(-1)), and maximal gastrin-stimulated acid secretion to 167.4 +/- 15.1 mumol h-1 with impromidine (10 mumol 1(-1)). Impromidine dose-dependently inhibited basal and gastrin-stimulated vascular histamine output. 4. The H3 receptor agonist R-a-methylhistamine, (1 and 10 mumol 1(-1)) minimally increased basal acid secretion. R-a-methylhistamine (10 mumol 1(-1)) did not significantly affect maximal gastrin-stimulated acid secretion. Basal and gastrin-stimulated vascular histamine outputs decreased to 4.0 +/- 0.8 (P less than 0.05) and 24.7 +/- 4.7 nmol h-1 (P = 0.05) with R-a-methylhistamine (10 mumol 1(-1)). 5. The H2 receptor antagonist ranitidine (2 mumol 1(-1)) did not inhibit basal acid secretion, but acid outputs with gastrin and all histamine agonists were reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absorption and presystemic glucuronidation of 1-naphthol in the vascularly fluorocarbon emulsion perfused rat small intestine

Summary Using the isolated vascularly fluorocarbon emulsion perfused rat small intestine some factors which determine the extent of the intestinal glucuronidation of 1-naphthol to 1-naphthol--d-glucuronide were studied. Increasing the luminal 1-naphthol concentration resulted in a concomitant increase in the 1-naphthol appearance in the vascular perfusate. In contrast, the total appearance of 1-naphthol--d-glucuronide increased less than proportional to the increase in the luminal 1-naphthol concentration. About 88% of the total amount of 1-naphthol--d-glucuronide excreted was released into the vascular perfusate. The capacity-limited intestinal glucuronide efflux is most likely due to saturation of the excretory mechanism for 1-naphthol--d-glucuronide. Decreasing the vascular flow rate influenced both the appearance of 1-naphthol and 1-naphtol--d-glucuronide in the vascular perfusate, whereas the appearance of 1-naphthol--d-glucuronide in the luminal perfusate was essentially flow-independent. A noradrenaline-induced change in the haemodynamic state of the vascular bed (with the total flow kept constant) resulted in a marked decrease in the 1-naphthol vascular concentration. The vascular 1-naphthol--d-glucuronide concentration was only slightly affected. These results indicate that changes in blood flow and blood flow distribution within the intestinal wall can affect the extent of presystemic intestinal metabolism by interfering with the absorption of the parent compound and the efflux of formed conjugates. These parameters can be of paramount importance for causing variable intestinal first-pass effects of drugs in vivo. Send offprint requests to M. H. de Vries at the above address