Calcium-free Environment Research Articles

Comparative evaluation of echocardiographic and morphometric characteristics of the rat heart left ventricle

Purpose of the study. Comparative evaluation of echocardiographic and morphometric dimensions of the rat heart left ventricle. Materials and methods. The study included 10 outbred male rats weighing 160–180 g. The size of the heart left ventricle was assessed using echocardiography and morphometry. The measurements were carried out according to standard protocols. To perform morphometric measurements, hearts were stopped in systole with a lethal dose (1.0 mg/kg) of 0.025 % strophanthin K solution, and in diastole, by immersing the hearts in a chilled physiological solution (calcium-free medium). Results. It was shown that the systolic size of the left ventricle of the heart according to echocardiography is 1.79Ѓ}0.10 mm, and morphometry 1.64Ѓ}0.09 mm (p = 0,302); diastolic size of the left ventricle, respectively, 3.42Ѓ}0.16 mm and 3.66Ѓ}0.17 mm (р = 0,318). The detected discrepancies do not exceed 10 % and, apparently, are due to the fact that the physiological dimensions of the heart left ventricle are measured by a non-invasive echocardiographic method, and for morphometric measurements of the dimensions in the systole, the heart was stopped by injection of strophanthin K, which entails an overload of heart cells with Ca2+ ions and as a result, contracture of cardiomyocytes; post-mortem cardiac arrest in diastole, in a calcium-free environment, is accompanied by a loss of cardiac muscle tone and, therefore, dilatation of the left ventricle will naturally be greater than physiological. Conclusion. Noninvasive echocardiographic measurement of the size of the heart left ventricle fully reflects the actual size of the left ventricle of the rat heart (the difference does not exceed 10 %), i. e. echocardiographic measurements are valid.

Motility disorders and disintegration into separate cells of Trichoplax sp. H2 in the presence of Zn2+ ions and L-cysteine molecules: A systems approach

Placozoa remain an ancient multicellular system with a dynamic body structure where calcium ions carry out a primary role in maintaining the integrity of the entire animal. Zinc ions can compete with calcium ions adsorption. We studied the effect of zinc ions and l-cysteine molecules on the interaction of Trichoplax sp. H2 cells. The regularity of formless motion was diminished in the presence of 20–25 μM of Zn2+ ions leading to the formation of branching animal forms. Locomotor ciliated cells moved chaotically and independently of each other leaving the Trichoplax body and opening a network of fiber cells. Application of 100 μM cysteine resulted in dissociation of the plate into separate cells. The combined chemical treatment shifted the effect in a random sample of animals toward disintegration, i.e. initially leading to disorder of collective cell movement and then to total body fragmentation. Two dissociation patterns of Trichoplax plate as “expanding ring” and “bicycle wheel” were revealed. Analysis of the interaction of Ca2+ and Zn2+ ions with cadherin showed that more than half (54%) of the amino acid residues with which Ca2+ and Zn2+ ions bind are common. The contact interaction of cells covered by the cadherin molecules is important for the coordinated movements of Trichoplax organism, while zinc ions are capable to break junctions between the cells. The involvement of other players, for example, l-cysteine in the regulation of Ca2+-dependent adhesion may be critical leading to the typical dissociation of Trichoplax body like in a calcium-free environment. A hypothesis about the essential role of calcium ions in the emergence of Metazoa ancestor is proposed.

The Effect of Plantain Active Ingredient Aucubin on Isolated Rat Smooth Muscle Tissue and Primary Cell Lines

This study aims to assess the pharmacodynamics of aucubin, the active ingredient in plantain (Plantago), on in vitro rat bladder and trachea tissues, and explore its cell protective effects on primary lung and kidney cell lines. The study was carried out via repeated applications of acetylcholine, atropine, verapamil and oxybutynin, alongside Ca++ in a calcium-free environment, on urinary bladder tissue, and repeated applications of acetylcholine, atropine, carbachol and mecamylamine on trachea tissue. At the same time, cell viability and catalase and superoxide dismutase activity was measured on primary cell lines. The results indicate that aucubin has a relaxant effect on urinary bladder and trachea tissues. It is conceived that aucubin acts as a cholinergic antagonist through different subreceptors (muscarinic-3 receptors). The results also indicate that aucubin has a dose-dependent effect on primary lung and kidney cell lines, with increased cell viability, reduced cytotoxicity and antioxidant effects. In conclusion, it is suggested that plantain, which is used in folk medicine, and its active ingredient aucubin might have beneficial effects on bronchoconstriction and other respiratory conditions, as well as on pulmonary and renal diseases, urinary incontinence, etc.

Missing elimination via membrane vesicle shedding contributes to the diminished calcium sensitivity of listeriolysin O

The lytic capacity of cholesterol-dependent cytolysins is enhanced in the extracellular calcium-free environment through a combination of limited membrane repair and diminished membrane toxin removal. For a typical neurotoxin of the group, pneumolysin, this effect has already been observed at reduced (1 mM) calcium conditions, which are pathophysiologically relevant. Here, we tested another neurotoxin of the group, listeriolysin O from L. monocytogenes, active in the primary vacuole after bacterium phagocytosis in host cells. Reduced calcium did not increase the lytic capacity of listeriolysin (in contrast to pneumolysin), while calcium-free conditions elevated it 2.5 times compared to 10 times for pneumolysin (at equivalent hemolytic capacities). To clarify these differences, we analyzed membrane vesicle shedding, known to be a calcium-dependent process for toxin removal from eukaryotic cell membranes. Both pneumolysin and listeriolysin initiated vesicle shedding, which was completely blocked by the lack of extracellular calcium. Lack of calcium, however, elevated the toxin load per a cell only for pneumolysin and not for listeriolysin. This result indicates that vesicle shedding does not play a role in the membrane removal of listeriolysin and outlines a major difference between it and other members of the CDC group. Furthermore, it provides new tools for studying membrane vesicle shedding.

Calcium-dependent disorder-to-order transitions are central to the secretion and folding of the CyaA toxin of Bordetella pertussis, the causative agent of whooping cough

The adenylate cyclase toxin (CyaA) plays an essential role in the early stages of respiratory tract colonization by Bordetella pertussis, the causative agent of whooping cough. Once secreted, CyaA invades eukaryotic cells, leading to cell death. The cell intoxication process involves a unique mechanism of translocation of the CyaA catalytic domain directly across the plasma membrane of the target cell. Herein, we review our recent results describing how calcium is involved in several steps of this intoxication process. In conditions mimicking the low calcium environment of the crowded bacterial cytosol, we show that the C-terminal, calcium-binding Repeat-in-ToXin (RTX) domain of CyaA, RD, is an extended, intrinsically disordered polypeptide chain with a significant level of local, secondary structure elements, appropriately sized for transport through the narrow channel of the secretion system. Upon secretion, the high calcium concentration in the extracellular milieu induces the refolding of RD, which likely acts as a scaffold to favor the refolding of the upstream domains of the full-length protein. Due to the presence of hydrophobic regions, CyaA is prone to aggregate into multimeric forms in vitro, in the absence of a chaotropic agent. We have recently defined the experimental conditions required for CyaA folding, comprising both calcium binding and molecular confinement. These parameters are critical for CyaA folding into a stable, monomeric and functional form. The monomeric, calcium-loaded (holo) toxin exhibits efficient liposome permeabilization and hemolytic activities in vitro, even in a fully calcium-free environment. By contrast, the toxin requires sub-millimolar calcium concentrations in solution to translocate its catalytic domain across the plasma membrane, indicating that free calcium in solution is actively involved in the CyaA toxin translocation process. Overall, this data demonstrates the remarkable adaptation of bacterial RTX toxins to the diversity of calcium concentrations it is exposed to in the successive environments encountered in the course of the intoxication process.

Stability, structural and functional properties of a monomeric, calcium\u2013loaded adenylate cyclase toxin, CyaA, from Bordetella pertussis

Bordetella pertussis, the causative agent of whooping cough, secretes an adenylate cyclase toxin, CyaA, which invades eukaryotic cells and alters their physiology by cAMP overproduction. Calcium is an essential cofactor of CyaA, as it is the case for most members of the Repeat-in-ToXins (RTX) family. We show that the calcium-bound, monomeric form of CyaA, hCyaAm, conserves its permeabilization and haemolytic activities, even in a fully calcium-free environment. In contrast, hCyaAm requires sub-millimolar calcium in solution for cell invasion, indicating that free calcium in solution is involved in the CyaA toxin translocation process. We further report the first in solution structural characterization of hCyaAm, as deduced from SAXS, mass spectrometry and hydrodynamic studies. We show that hCyaAm adopts a compact and stable state that can transiently conserve its conformation even in a fully calcium-free environment. Our results therefore suggest that in hCyaAm, the C-terminal RTX-domain is stabilized in a high-affinity calcium-binding state by the N-terminal domains while, conversely, calcium binding to the C-terminal RTX-domain strongly stabilizes the N-terminal regions. Hence, the different regions of hCyaAm appear tightly connected, leading to stabilization effects between domains. The hysteretic behaviour of CyaA in response to calcium is likely shared by other RTX cytolysins.

Agonist-induced sensitisation of the irritant receptor ion channel TRPA1.

The transient receptor potential ankyrin 1 (TRPA1) ion channel is expressed in nociceptive neurons and its activation causes ongoing pain and inflammation; TRPA1 is thought to play an important role in inflammation in the airways. TRPA1 is sensitised by repeated stimulation with chemical agonists in a calcium-free environment and this sensitisation is very long lasting following agonist removal. We show that agonist-induced sensitisation is independent of the agonist's binding site and is also independent of ion channel trafficking or of other typical signalling pathways. We find that sensitisation is intrinsic to the TRPA1 protein and is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials. Agonist-induced sensitisation may provide an explanation for sensitisation following long-term exposure to harmful irritants and pollutants, particularly in the airways. The TRPA1 ion channel is expressed in nociceptive (pain-sensitive) neurons and responds to a wide variety of chemical irritants, such as acrolein in smoke or isothiocyanates in mustard. Here we show that in the absence of extracellular calcium the current passing through TRPA1 gradually increases (sensitises) during prolonged application of agonists. Activation by an agonist is essential, because activation of TRPA1 by membrane depolarisation did not cause sensitisation. Sensitisation is independent of the site of action of the agonist, because covalent and non-covalent agonists were equally effective, and is long lasting following agonist removal. Mutating N-terminal cysteines, the target of covalent agonists, did not affect sensitisation by the non-covalent agonist carvacrol, which activates by binding to a different site. Sensitisation is unaffected by agents blocking ion channel trafficking or by block of signalling pathways involving ATP, protein kinase A or the formation of lipid rafts, and does not require ion flux through the channel. Examination of the voltage dependence of TRPA1 activation shows that sensitisation is accompanied by a slowly developing shift in the voltage dependence of TRPA1 towards more negative membrane potentials, and is therefore intrinsic to the TRPA1 channel. Sensitisation may play a role in exacerbating the pain caused by prolonged activation of TRPA1.

A novel technique using hydrophilic polymers to promote axonal fusion.

The management of traumatic peripheral nerve injury remains a considerable concern for clinicians. With minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury, outcomes of surgical intervention have been unpredictable. The inability to manipulate the pathophysiology of nerve injury (i.e., Wallerian degeneration) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration (~1 mm/day). When axons are severed, the endings undergo calcium-mediated plasmalemmal sealing, which limits the ability of the axon to be primarily repaired. Polythethylene glycol (PEG) in combination with a bioengineered process overcomes the inability to fuse axons. The mechanism for PEG axonal fusion is not clearly understood, but multiple studies have shown that a providing a calcium-free environment is essential to the process known as PEG fusion. The proposed mechanism is PEG-induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur. This review highlights PEG fusion, its past and current studies, and future directions in PEG fusion.

Poloxamer 188 reduces the contraction-induced force decline in lumbrical muscles from mdx mice

Duchenne Muscular Dystrophy is a genetic disease caused by the lack of the protein dystrophin. Dystrophic muscles are highly susceptible to contraction-induced injury, and following contractile activity, have disrupted plasma membranes that allow leakage of calcium ions into muscle fibers. Because of the direct relationship between increased intracellular calcium concentration and muscle dysfunction, therapeutic outcomes may be achieved through the identification and restriction of calcium influx pathways. Our purpose was to determine the contribution of sarcolemmal lesions to the force deficits caused by contraction-induced injury in dystrophic skeletal muscles. Using isolated lumbrical muscles from dystrophic (mdx) mice, we demonstrate for the first time that poloxamer 188 (P188), a membrane-sealing poloxamer, is effective in reducing the force deficit in a whole mdx skeletal muscle. A reduction in force deficit was also observed in mdx muscles that were exposed to a calcium-free environment. These results, coupled with previous observations of calcium entry into mdx muscle fibers during a similar contraction protocol, support the interpretation that extracellular calcium enters through sarcolemmal lesions and contributes to the force deficit observed in mdx muscles. The results provide a basis for potential therapeutic strategies directed at membrane stabilization of dystrophin-deficient skeletal muscle fibers.

Temperature-dependent inhibition of motility in spermatozoa from different avian species.

This study demonstrates that the pattern of temperature-dependent inhibition of chicken sperm motility at 40 degrees C in vitro, and its release by calcium, is also found in drake spermatozoa and, partially, in turkey spermatozoa. However, no such temperature-dependent inhibition was found in spermatozoa from Japanese quail and Houbara bustard, for which physiological levels of calcium at 40 degrees C had an inhibitory and no effect on sperm motility, respectively. Thus, on the basis of this evidence on the regulation of avian sperm motility in vitro, the hypothesis that oviducal sperm storage tubules might immobilise spermatozoa by providing a calcium-free environment in vivo does not appear to be universally applicable to all species of birds.

Regulation of M current by intracellular calcium in bullfrog sympathetic ganglion neurons

Regulation of M current (lM) by intracellular free calcium was studied in dissociated bullfrog sympathetic ganglion B cells using whole-cell recording, intracellular perfusion, and confocal calcium imaging. BAPTA (20 mM) and appropriate amounts of calcium were added to pipette solutions to clamp calcium at different levels. A high concentration of BAPTA itself mildly inhibited lM. Intracellular perfusion effectively controlled cellular free calcium; this was confirmed by confocal imaging with the calcium indicator fluo-3. In a calcium-free environment (no calcium added to either side of the cell membrane), average lM was 166 pA. Raising intracellular free calcium to 60 nM or higher reversibly enhanced lM by more than 100%. The maximum M conductance doubled upon raising calcium from 0 to 120 nM, and was accompanied by a -11 mV shift of the half-activation voltage. The kinetics of the closing and reopening relaxations of lM were also altered by raising calcium. Enhancement of lM by calcium required ATP in the pipette. TEA (5 mM) and d-tubocurarine (d-TC; 100 microM) did not alter the calcium effect, indicating that it was the M current being modulated and not other K+ currents. High calcium (450 nM) reduced lM. The up- and downregulation of lM paralleled the increases and decreases of fluorescence intensity observed via calcium imaging. Changing extracellular calcium had no significant effect on lM or cellular fluorescence. The role of calcium in muscarinic and peptidergic modulation of lM was also explored. Muscarine (1 or 10 microM) inhibited lM less at zero calcium than at higher calcium. Nearly complete suppression occurred with 120 nM calcium in the presence of 20 mM BAPTA. lM overrecovered upon washout of muscarine at 120 nM calcium, while little overrecovery of lM developed at zero calcium. Similar effects were observed at zero and 120 nM calcium when using the peptide LHRH to inhibit lM. We conclude that the absolute level of free calcium determines the size of lM, and that a minimum sustained level of calcium is required both for optimal suppression of lM by muscarine and for overrecovery. While our data suggest that resting calcium levels play a permissive role in muscarinic suppression, an additional role for agonist-induced calcium increases cannot be ruled out.

Role of calcium in monitor peptide-stimulated cholecystokinin release from perifused intestinal cells.

Monitor peptide stimulates cholecystokinin (CCK) release from the intestine, but the cellular mechanisms responsible for this effect are uncertain. In the present study, the roles of membrane potential difference and calcium influx in monitor peptide-mediated CCK release were examined in a perifusion system containing isolated mucosal cells from the rat duodenum. This method represents an in vitro system in which CCK-releasing cells can be challenged with secretagogues or other maneuvers to study the dynamics of hormone secretion. High concentrations of KCl (50 mM), which reduce electrical potential difference across the cell membrane, caused the release of CCK. This effect was inhibited by the calcium channel blocker MnCl2. Monitor peptide stimulated CCK release in a dose-dependent manner at concentrations from 3 x 10(-12) to 3 x 10(-8) M. The requirement for extracellular calcium in secretagogue-stimulated release of CCK was investigated using ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), a calcium chelator, and MnCl2. A calcium-free environment supplemented with 2 mM EGTA completely inhibited CCK secretion in response to stimulatory doses of monitor peptide. CCK secretion was restored when calcium was reintroduced into the system. Similarly, MnCl2 completely blocked monitor peptide-stimulated CCK release. These data indicate that membrane depolarization and monitor peptide stimulate the release of CCK through calcium-dependent mechanisms, suggesting that increases in intracellular calcium within CCK cells are likely to be important in CCK release.

Cocaine: An in vivo microdialysis evaluation of its acute action on dopamine transmission in rat striatum

The effects of cocaine on dopamine (DA) neurotransmission were evaluated by in vivo microdialysis in the striatum of halothane-anesthetized rats. Intravenous cocaine produced a dose-dependent, transient increase of the extracellular concentration of DA, with a peak response within 10 min and a return to control level by 30 min. The sharp DA response pattern was abolished in a calcium-free environment, indicating that DA release enhanced by cocaine originates from a vesicular storage pool. Continuous administration of cocaine (via the perfusion medium) directly into the nigrostriatal terminal region also produced a dose-dependent increase in DA release. Low concentrations (10(-5) M and 10(-6) M) of cocaine maintained DA at a constant stable level, consistent with the effects observed after potent DA uptake inhibitory agents (e.g., nomifensine and Lu19005). However, continuous exposure to high concentrations (greater than or equal to 10(-4) M) induced a transient elevation of DA within 20 min, following which DA decreased to a stable but high level; this decrease might reflect tolerance to the effect of cocaine. Administration of cocaine (10(-3) M) into the substantia nigra did not change striatal DA release. The local striatal action of cocaine was less potent than amphetamine in elevating DA overflow and in its effect on DA metabolism. These findings suggest that the fast transient enhancement of DA by intravenous cocaine is most likely a consequence of the transient presence of cocaine in the terminal region, correlating with the well-known rapid pharmacokinetic and behavioral aspects of the drug.

Cell injury of cultured rat myocardial cells after reoxygenation of hypoxic cultures in the presence and absence of calcium.

Primary cultures of rat myocytes were deprived of oxygen (approximately 5 mm Hg, pO2) for 2 h in the presence or absence of calcium and were subsequently incubated for different time periods under normoxic (approximately 120 mm Hg, pO2) conditions. Myocyte calcium content was determined at the end of the oxygen-free period and after repletion of oxygen. Lactate dehydrogenase (LDH) release from the cells into the medium was used as an index of cell injury. Cultures deprived of oxygen in the absence of calcium showed a significant decrease in myocyte calcium content at the end of the oxygen-free period. However, the calcium content of myocyte cultures deprived of oxygen in the presence of calcium did not change significantly. The enzyme release of both groups of oxygen-deprived cultures was similar to that of controls. A progressive increase in myocyte calcium content was observed 5, 10, 15, and 30 min after repletion of oxygen in both groups. Reoxygenation of cultures subjected to hypoxia in the presence of calcium caused minimal LDH leakage. However, major enzyme release was observed 30 min after oxygen repletion in cultures previously subjected to hypoxia in a calcium-free environment. The present study shows that reoxygenation injury is more severe in myocyte cultures previously deprived of both oxygen and calcium than in cultures deprived of oxygen in the presence of calcium.

Rhythmic electrical activity in rabbit aorta induced by EGTA.

Normal electrical activity in smooth muscle is dependent on the presence of calcium in the extracellular fluid (Bennett, 1967; Brading, Bülbring & Tomita, 1969; Mangel & Prosser, 1980; Weigel, Connor & Prosser, 1979). However, it has recently been demonstrated that calcium-free solutions containing EGTA induce two types of spontaneous electrical activity in visceral smooth muscle. Prolonged depolarizations of about 17 s were recorded under these conditions from segments of cat small intestine and the stomach of the skate, toad or frog (Prosser et al. 1977), while rat small intestine produced fast electrical potentials of approximately 0·85 s duration (Mangel & Nelson, 1978). Since both potential types were eliminated by removal of external sodium or by addition of calcium channel blockers, it was concluded that their mechanism of generation consisted of sodium ions traversing channels normally used by calcium. In this report we examine whether EGTA is capable of inducing electrical activity in a vascular smooth muscle which does not exhibit spontaneous activity in normal saline.Electrical activity was recorded with pressure electrodes (Bortoff, 1961) from segments of aorta removed from rabbits killed by concussion. The muscle segments were placed immediately into Krebs saline of the following composition (mm): NaCl, 118·5; KC1, 4·7; MgCl2, 1·2; NaHCO2, 23·8; KH2PO4, 1·2; CaCl2, 2·5; and glucose, 5·5. The calcium-free saline used in this study had no added calcium plus 3 mm EGTA. Normal Krebs saline and calcium-free Krebs saline were aerated with a 95 %o2–5% CO2 gas mixture. When manganese was tested, the saline was buffered with MOPS. MOPS saline was as follows (mm): NaCl, 142·3; KC1, 5·9; CaCl2, 2·5; MgCl2, 1·2; glucose, 5·5 and MOPS, 4·0. This solution was bubbled with 100&% O2. All experiments were at 35–37 &°C.Electrical recordings made from rabbit aorta in normal saline showed no spontaneous electrical activity (Fig. 1 A). Following a 21 min incubation period in calcium-free saline, fast rhythmic potentials developed (Figs. 1B, C). In other preparations incubation period between 11–72 min was necessary before the initiation of nativity. The mean duration of the fast potentials was 1·02±0·01 s. (n = 38) a range from 0·55 to 2·5 sec5 sec. Typically, there was considerable variation in the wave form and duration of fast potentials within a single preparation. In several cases a slow depolarization, similar to intestinal prepotentials (Brading et al. 1969; Liu, Prosser & Job, 1969), preceded each fast potential. Fast potentials were often superimposed upon a slower underlying rhythm (Fig. 1B). There were no consistent changes observed in fast potential activity during the rising or falling phase of the slower rhythm. No rhythmic mechanical activity was found to be associated with fast potentials as has been shown to correspond to prolonged electrical potentials in cat small intestine (Mangel et al. 1979).The ionic mechanism responsible for generation of fast potentials was studied next. Replacement of saline NaCl with LiCl resulted in reversible elimination of fast potentials (Fig. 2). However, in the presence of 5 μM tetrodotoxin or 100 μM ouabain activity persisted. Following the addition of calcium channel blockers (manganese, D600) activity was eliminated within 3 min (see Fig. 3). Reduction in external magnesium concentrations did not result in elimination of fast potentials.In the calcium-free saline used in this study, an inward-directed calcium gradient would not exist. Therefore, fast potentials cannot result from an influx of calcium. Elimination of fast potentials in sodium-deficient saline suggests that sodium ions are the current carriers responsible for fast potential generation. From the failure of tetrodotoxin to eliminate fast potentials, along with their sensitivity to calcium channel blockers, the most probable mechanism responsible for the production of fast potentials is sodium ions traversing calcium channels.Prosser et al. (1977) concluded that in preparations which show spontaneous activity in normal saline, EGTA induction of rhythmic electrical activity is a general property of smooth muscle membranes. We have extended that observation by showing that rabbit aorta, a preparation quiescent in normal saline, will also produce rhythm activity following EGTA treatment. Under physiological conditions, smooth muscle membranes will not be exposed to a calcium-free environment; thus, fast potentials would not be expected to be present. However, from the present study, it can be concluded that the cellular machinery to generate rhythmic electrical activity is inherent in smooth muscle membranes. Under in vivo conditions where neural connections are present recordings from the aorta may show rhythmic activity.This research was supported by American Heart Association grant no. 781124, NIH grant no. HL 07188 and American Heart Association Fellowship no. RF-1. Special thanks to Barbara Bradie for typing the manuscript.

A COMPARISON OF THE DIFFERENTIAL EFFECTS OF NITROGLYCERIN, NIFEDIPINE AND PAPAVERINE ON CONTRACTURES INDUCED IN VASCULAR AND INTESTINAL SMOOTH MUSCLE BY POTASSIUM AND LANTHANUM

AbstractTo elucidate the mechanisms of smooth muscle relaxant effects of nitroglycerin, the effects of this substance on the potassium contracture (K+−contracture) and the lanthanum-induced contracture (La3+−contracture) were studied using smooth muscle preparations of the canine coronary artery and colon and the findings compared with the effects of nifedipine, a representative calcium antagonistic vasodilator. The effects of papaverine, a prototype smooth muscle relaxant, were also studied. La3+−contracture was induced in a calcium-free environment with the addition of lanthanum, an effective blocker of calcium influx. In the coronary artery, nitroglycerin produced a relaxation both of the La3+−contracture and the K+−contracture. However, neither La3+−contracture nor K-contracture in the colon was affected. Nifedipine did not relax the La3+−contracture in a range of doses at which K+−contracture of both types of smooth muscles was relaxed. Papaverine produced a relaxation of La3+−contracture as well as K+−contracture, in both types of smooth muscles. Unlike the mechanism related to the relaxant effects of nifedipine, which is generally admitted to be an inhibition of calcium influx, the relaxant effect of nitroglycerin was attributed to the suppression of calcium release from the intracellular store sites and/or stimulation of calcium uptake into the intracellular store-sites. Papaverine was assumed to produce a relaxation through augmentation of the calcium binding to the intracellular store sites for calcium as well as through inhibition of the calcium influx.

Electrical activity of lymphatic smooth muscles.

SummaryMembrane action potentials of bovine mesenteric lymphatics were recorded simultaneously with isometric contractions by the use of the sucrose gap method. The action potentials, about 3 sec in duration, always had one-to-one correspondence to the contraction waves. Each action potential consisted of three phases, i.e., initial slow depolarization, spike, and slow repolarization. The application of nor-adrenalin elicited a slight but long-lasting depolarization superimposed by frequent discharges of action potentials. The administration of acetylcholine in a relatively high concentration to the preparations induced a transient depolarization followed by the occurrence of action potentials. From the effects of tetrodotoxin, manganese, calcium-free environment, and barium chloride on the spontaneous contractions, it was suggested that calcium current may probably play a major role in producing spike discharge in bovine lymphatic smooth muscles.

The effects of prolactin, cortisol and calcium-free environment on water influx in isolated gills of Japanese eel, Anguilla japonica

1. The effects of prolactin, cortisol and a calcium-free environment on water influx into the isolated gills of the Japanese eel, Anguilla japonica were studied by an in vitro technique. 2. It was confirmed that the prolactin injection significantly reduced the water influx into the isolated gills and/or water permeability. Cortisol injection, on the other hand, increased the water influx and/or water permeability. 3. Calcium-free environments, deionized water and calcium-free sea water, served to increase the water influx and/or water permeability compared with its values in normal environments. 4. The possible roles of prolactin, cortisol and environmental calcium in the osmoregulation of the eel in fresh water and sea water are discussed.

Actions of calcium and certain multivalent cations on potassium contracture of guinea-pig's taenia coli.

1. Using a calcium buffer system, the effect of severe calcium lack on the shape of K-contracture of the guinea-pig's taenia coli was studied. Under conditions of calcium lack, the initial phasic response was preferentially affected and it disappeared completely at concentrations below 10(-7)M, while the ensuing tonic response persisted, though considerably diminished in size, even at the concentration of 10(-8)M.2. In calcium-free media, various multivalent cations, which according to Frank (1962) can support the K-contracture of a skeletal twitch muscle fibre in calcium-free solution, augmented the remaining tonic response, but did not restore the phasic response, when it was eliminated in calcium free environment.3. When K-contractures were induced in normal calcium media, these cations produced, in contrast, an abolition of the phasic response together with a partial depression of the tonic phase. They also inhibited a part of the fully developed contracture. The last effect was no longer obtainable in calcium-free media.4. It is concluded that the phasic response and a part of the tonic response of taenia coli depend upon the extracellular calcium for their initiation (and also for maintenance of tension in the case of the latter) and that the rest of the tonic response draws on a store of ;bound' calcium for its evolution.