Estimation of the levels of calcium and other electrolytes during various phases of contraction of the longitudinal smooth muscle of the guinea pig ileum

A method is described for measuring calcium and other electrolyte levels during various phases of contraction of the longitudinal smooth muscle of the guinea pig ileum. Muscles are immersed in a solution of 160 mM Tris-Cl containing 10 mM lanthanum for 30 min to reduce the contribution of extracellularly bound cations, while the temperature is reduced to 4°C to prevent active cation fluxes. The muscle cells gained calcium and lost magnesium during the phasic and tonic phases of contractions by the muscarinic agent, CD (cis-2-methyl-4-dimethylaminomethyl-1, 3-dioxolane methiodide) and during the tonic phase of contractions by KCl. Muscles lost potassium during contractions to CD, but gained potassium during contractions to 60 mM KCl. The potassium lost during contractions to CD was regained slowly over a period of 30 min when the CD was washed out. This recovery corresponded to the rate of recovery of spontaneous activity and normal responsiveness of the muscle to further doses of stimulants. Calcium levels were reduced rapidly to below normal levels on washout of the CD and were regained in a similar time course to that of potassium. Sodium levels were not significantly changed with either stimulant.     

Monoamine and diamine oxidative deamination in the longitudinal smooth muscle of guinea pig ileum

The longitudinal smooth muscle of guinea pig ileum contains three different types of oxidative deaminating enzymes: monoamine oxidase types A and B, diamine oxidase and a soluble clorgyline-deprenyl-resistant benzylamine oxidase. These enzymes have different subcellular locations. The longitudinal smooth muscle of guinea pig ileum oxidatively deaminates beta-phenylethylamine at a much higher rate than benzylamine. beta-Phenylethylamine is a good substrate for monoamine oxidase type B but also for the soluble clorgyline-deprenyl-resistant benzylamine oxidase. On the other hand, benzylamine is oxidised by mitochondrial monoamine oxidase, by the clorgyline-deprenyl-resistant enzyme and by diamine oxidase.     

Opioids modulate periodicity rather than efficacy of peristaltic waves in the guinea pig ileum in vitro

The influence of opioid receptor blockade by naloxone and opioid receptor activation by opioids on peristalsis was studied in isolated segments of the guinea pig ileum.1. (-)Naloxone, but not (+)naloxone, increased the mean number of peristaltic waves per min within periods of elevated intraluminal pressure. Naloxone tended to modify intermittent peristalsis into ongoing peristalsis, whereas opioids worked in an opposite fashion. 2. Maximum amplitudes of luminal volume displacement during single peristaltic waves were not decreased by opioids. (-)Naloxone, however, applied to non-pretreated segments, decreased transitorily the efficacy of single peristaltic waves to a small, but statistically significant degree 3. Enhancement of peristalsis by naloxone decreased over time, although enough naloxone was present to occupy all opioid receptors. This suggests that opioid receptor blockade induces some compensatory mechanism.     

Activation of protein kinase in the guinea pig fundic gastric mucosa by histamine.

The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa. Histamine induced a significant elevation of tissue cyclic AMP levels and also $\text{in situ}$ activation of the protein kinase. 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these two parameters. Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination. It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplastic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form. The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.     

Cholinesterases and choline acetyltransferase in the longitudinal muscle of the guinea pig ileum

Summary In order to gain insight into the possible role of the ACh-system in the smooth muscle cell, the presence of choline acetyltransferase, acetylcholinesterase and butyrylcholinesterase was studied in the longitudinal muscle of the guinea-pig ileum after the mechanical removal of Auerbach's plexus. Such treatment completely removes all nerve elements as confirmed by histochemistry and electron-microscopic examination.It was found that in the longitudinal muscle devoid of all nervous elements a substantial percentage of the activity of all three enzymes still remained. Ultrastructural localization of acetylcholinesterase and butyrylcholinesterase was observed on the sarcolemma, sarcoplasmic reticulum, nuclear membrane and invaginations of the sarcolemma. The localization of cholinesterases coincides with sites which are presumably involved in calcium movements during contraction and relaxation. It is well known that the depolarized smooth muscle responds to exogenous ACh with a reversible, calcium dependent contraction and it was suggested that ACh may act by increasing the influx of calcium through the cell membrane or by liberating calcium from its bound form.The presence of choline acetyltransferase and cholinesterase activities in the muscle cell proper, as well as the localization of cholinesterases on structures connected with calcium movements, support the coexistence of an intrinsic cholinergic mechanism in the smooth muscle.Part of this work was presented at the 6^th International Meeting of the International Society for Neurochemistry, Copenhagen, 1977     

Relationship between stimulated phosphatidic acid production and inositol lipid hydrolysis in intestinal longitudinal smooth muscle from guinea pig.

Accumulation of [32P]phosphatidic acid (PA) and total [3H]inositol phosphates (IPs) was measured in the longitudinal smooth-muscle layer from guinea-pig small intestine. Stimulation with carbachol, histamine and substance P produced increases in accumulation of both [3H]IPs and [32P]PA over the same concentration range. The increase in [32P]PA accumulation in response to carbachol (1 microM-0.1 mM) was inhibited in the presence of atropine (0.5 microM). Buffering the external free [Ca2+] to 10 nM did not prevent the carbachol-stimulated increase in [32P]PA accumulation. Carbachol and Ca2+ appear to act synergistically to increase accumulation of [32P]PA. In contrast, although incubation with noradrenaline also increased accumulation of [3H]IPs, no increase in accumulation of [32P]PA could be detected. These results suggest that an increase in formation of IPs is not necessarily accompanied by an increase in PA formation, and imply the existence of receptor-modulated pathways regulating PA concentrations other than by phospholipase-C-catalysed inositol phospholipid hydrolysis.     

Phospholipid metabolism in acetylcholine and substance P induced desensitization of guinea pig ileum smooth muscle

The amount and the rate of inorganic phosphate incorporation into mono-, di- and triphosphoinositides and into total phospholipids of ileum smooth muscle were studied at various time of exposure to physiological concentrations of acetylcholine and substance P. The data obtained show a phase character of the mediator influence on the phosphate incorporation into mono and triphosphoinositides-a stimulation initial period, an inhibition during the desensitization period and a return to the normal level after the mediator elimination.     

A new prostaglandin E1 analogue (CL115, 574): I. Antisecretory and cytoprotectige effects in the rat

The effect of a new analogue of PGE₁ (CL115, 574) on gastric acid secretion, mucus secretion and protection against stress and indomethacin- induced ulcers were studied in the rat. CL115, 574 was more potent than PGE₁ and cimetidine in inhibiting acid secretion. CL115, 574 protected against the development of stress and indomethacin-induced ulcers prevented the indomethacin-induced decrease in hematocrit at an ED₅₀ (3 μg/kg) far below the antisecretory ED₅₀ (1 mg/kg). While the inhibiting acid secretion, CL115, 574 increased the volume of gastric secretion indicating a stimulation of nonparallel cell secretion by the rat stomach. In addition the compound stimulated the secretion of mucus into the gastric juice. On the basis of its potency as an inhibitor of acid secretion and these additional effects which are indicative of cytoprotective activity, CL115, 574 should be further studied as a possible anti-ulcer agent in man.     

Comparative anticholinergic potencies of R- and S- disopyramide in longitudinal muscle strips from guinea pig ileum

The anticholinergic potencies of R- and S- disopyramide were studied in isolated myenteric plexus longitudinal muscle strip preparations from guinea-pig ileum using two experimental procedures. The first procedure tested the relative potencies of the isomers in inhibiting electrically-stimulated contractions in 6 ileum preparations. A balanced crossover design was employed. The mean concentration of S-disopyramide required to inhibit electrically stimulated contractions by 50% was 4.6 × 10^?6 M and was about one-fourth of the concentration of R-disopyramide required to produce the same effect (p < 0.05). The second procedure tested the relative potencies of the isomers as direct antagonists of contractions induced by either histamine or acetylcholine in ileum preparations. Neither isomer antagonized the histamine-induced contractions. For the contractions induced by acetylcholine, the pA₂ value, obtained directly from Schild plots, was 6.25 for S- and 5.74 for R- disopyramide. However, the slope of the Schild plot for the S-isomer differed significantly from ?1, suggesting that other mechanisms in addition to direct antagonism of acetylcholine may be involved. Thus, the results of the experiments involving both the antagonism of electrically stimulated contractions and the direct antagonism of acetylcholine-induced contractions indicate (1) that both isomers of disopyramide have anticholinergic properties and (2) that S-disopyramide is about 3–4 fold more potent as an anticholinergic agent.     

Differential effects of TAK-802, a selective acetylcholinesterase inhibitor, and carbamate acetylcholinesterase inhibitors on contraction of the detrusor smooth muscle of the guinea pig

The aim of this study was to compare the effects of TAK-802, a novel acetylcholinesterase (AChE) inhibitor, and carbamate AChE inhibitors on the detrusor smooth muscle contractility in vitro using isometric tension measurements. The effects of drugs on the nicotine-induced contractions and basal tone of the isolated detrusor muscle of the guinea pig were examined. All of the drugs, namely, TAK-802, distigmine, neostigmine and pyridostigmine, enhanced the nicotine-induced contractions of the muscle strips in a concentration-dependent manner. On the other hand, while neostigmine and pyridostigmine markedly increased the basal tone, and distigmine slightly but significantly increased the basal tone, TAK-802 had no influence on the basal tone of the muscle strips at all. However, following co-treatment with tetraisopropyl pyrophosphoramide, a selective butyrylcholinesterase (BuChE) inhibitor, TAK-802 also did increase the basal tone. The increase of the basal tone by all of the above treatments was completely abolished by atropine. These results reveal that while all the four AChE inhibitors enhanced endogenous acetylcholine-induced contractions, their effects on the basal tone were clearly different. The effect of carbamate AChE inhibitors of increasing the basal tone could be partly attributed to their dual inhibition of both AChE and BuChE, because both cholinesterases may play a critical role in maintaining the resting tension of the urinary bladder. TAK-802, however, did not increase the basal tone of the detrusor muscle strips, probably because of its selective inhibitory effect against AChE. The effect of carbamate AChE inhibitors on the basal tone of the detrusor muscle may explain the decrease of bladder compliance observed in our previous study on guinea pigs as well as the deterioration of the bladder-storage function reported with their clinical use.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

The influence of harmaline on the movements of sodium ions in smooth muscle of the guinea pig ileum

Summary The effect of changing the extracellular concentration of both Na⁺ and K⁺ on the longitudinal muscle of the guinea-pig ileum was studied in the presence and absence of harmaline. A decrease in extracellular Na⁺ concentration was found to produce a dose-dependent contractile response, which may suggest the existence of a Na...Ca exchange mechanism in this muscle. Harmaline (2 × 10⁻⁴ M) was found to reversibly inhibit this contraction and was also found to selectively block the tonic component of high-K induced contradictions. In view of the fact that harmaline is a non-competitive inhibitor of Ca-induced contractions (Hider et al., Europ. J. Pharmacol., 71, 87, 1981), the action of harmaline was interpreted as being a specific inhibitor of the Na... exchange mechanism, binding specifically to Na⁺ coordination sites.     

The effects of a macrophage-derived cytotoxin on the growth and metabolism of target cells.

Peritoneal macrophages obtained from Sarcoma I (SaI)-immune CS7BL/6 mice release a heat-labile cytotoxin, specific macrophage cytotoxin (SMC), following a two hour interaction with appropriate target cells. Specific macrophage cytotoxin specifically inhibits A/Jax spleen cells from mitogenically responding to concanavalin A, whereas syngeneic CS7BL/6 spleen cells are unaffected. Treatment of target cells with SMC results in early alterations in RNA and DNA metabolism. The uptake and incorporation of ³H-uridine was found to be initially elevated while intracellular levels of ³H-thymidine became markedly reduced. Furthermore, the cytotoxic action of SMC was found to be rapidly accelerated and amplified by low levels of actinomycin-D.     

Membrane mechanisms of activating depolarized smooth muscle contraction (guinea pig small intestine) during exposure to physiologically active compounds

The experiments were performed on the depolarized smooth muscle of taenia coli with the use of double sucrose-gap arrangement. Muscle contractions were induced by 1) application of hystamin or bradikynin; 2) rectangular long-lasting (10--20 s) pulses of hyperpolarizing current--the strong contraction appeared in response to the switching off the current (off-response). Both the on- and off-responses to the hyperpolarizing current recorded before, during and after hystamine (or bradikynin)-induced contration were, as a rule, very similar. Treatment of smooth muscle with local anaesthetics (procaine, trimecaine, QX-572) removed hystamine- and bradikynin-induced contrations and only decreased off-responses. The analysis of the data obtained suggested the existence of the independent electrically and chemically excitable systems (channels?) OF Ca2+ ion transport in the membrane of smooth muscle cells.     

Comparison of muscarinic and alpha-adrenergic receptors in rat atria based on phosphoinositide turnover

The effects of muscarinic and alpha-adrenergic receptor stimulation on phosphoinositide turnover in rat atria have been compared. Despite the similar densities of muscarinic receptors in rat left and right atria, 0.1 mM carbachol increased [32P]phosphate incorporation into phosphatidylinositol (PI) by 35% (p less than 0.05) in left atria but had no effect in right atria. By contrast to the small muscarinic receptor effect, stimulation of alpha 1-adrenergic receptors by 0.1 mM methoxamine produced a more than two fold increase in [32P]phosphate incorporation into PI in both left and right atria, despite the reported smaller density of alpha-adrenergic receptors in rat atria compared to muscarinic receptors. Enhanced phosphate labelling by methoxamine did not occur in phospholipids other than PI, and was blocked by the alpha-adrenergic antagonist, phentolamine (20 microM). The results indicate that the majority of the muscarinic receptors in rat atria are not coupled to phosphoinositide turnover. If indeed the observed enhancement in [32P]-phosphate labelling by carbachol reflects phosphoinositide turnover, and assuming equal coupling efficiencies of muscarinic and adrenergic receptors, it is calculated that not more than 2% of the muscarinic receptors in rat left atria are coupled to this response.     

Effects of prostaglandin E2 (PGE2) on estradiol-17β-induced luteolysis in the nonpregnant ewe

Fifteen ewes were assigned as they came into estrus to the following randomized treatment groups: 1) Vehicle (1 ml corn oil + vehicle Na₂CO₃ buffer), 2) Estradiol-17β + vehicle and 3) Estradiol-17β + PGE₂ (500 μg) in Na₂CO₃ buffer (5 ewes/treatment group). Prostaglandin E₂ was given through an intrauterine cannula every four hours from days 8 through 15 postestrus. PGE₂ prevented a luteolytic dose of estradiol-17β given on days 9 and 10 from causing a precious luteolysis. PGE₂ maintained concentrations of progesterone in peripheral blood (days 8 through 15) and weights and concentrations of progesterone in corpora lutea on day 15 postestrus of ewes receiving estradiol-17β. It is concluded that chronic intrauterine infusions of PGE₂ can prevent an estradiol-17β-induced premature luteolysis.