Tension Of Strips Research Articles

Role of intracellular pH in relaxation of porcine tracheal smooth muscle by respiratory gases.

Hypercapnia and hypoxia both relax airway smooth muscle, but the mechanisms responsible are poorly understood. Because hypercapnia and hypoxia can each decrease intracellular pH (pHi) and acidosis can inhibit Ca2+ channels, we hypothesized that decreased pHi mediates relaxation of trachealis muscle by each of these respiratory gases. To examine the relationship between pHi and tone, we measured isometric tension, bath pH, and fluorescence intensity (540 nm) in porcine tracheal smooth muscle strips loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and excited alternately with 440- and 500-nm light. Strips equilibrated in Krebs-Henseleit solution bubbled with 95% O2-5% CO2 were contracted with carbachol and then relaxed with either 95% N2-5% CO2 or 93% O2-7% CO2. The ratio of fluorescence intensity at 500 nm to 440 nm was calibrated vs. pHi with use of nigericin. Baseline pHi was 7.19 +/- 0.03 (n = 13). Hypoxia decreased active tension by approximately 60% but did not change pHi. Hypercapnia induced decreases in tension that were associated with substantial decreases in pHi. Thus, decreased pHi does not mediate hypoxic relaxation, but the relaxation during physiologically relevant increases in CO2 concentration is associated with significant cellular acidification.

The effects of extracellular pH on intracellular pH, Ca2+ and tension of canine tracheal smooth muscle strips

The effects of changes in extracellular pH (pHo) on intracellular Ca2+ concentration ([Ca2+]i) or intracellular pH (pHi) were measured simultaneously with muscle tension in canine tracheal smooth muscle strips. [Ca2+]i and pHi were measured using the fluorescent dyes fura-2 and BCECF, respectively. During high K(+)-induced contractions (24.2, 36.4 or 72.7 mM) at pH 7.4, pHo was changed to 7.8 or 7.0 with NaOH or HCl, respectively. Induced changes in pHi were equal to approximately 50% of the changes in pHo. Alkalinization significantly increased [Ca2+]i and enhanced muscle contraction at all concentrations of K+ but did not alter the relationship between muscle tension and [Ca2+]i. Acidification significantly decreased [Ca2+]i without changing muscle tone; hence, the muscle tension-[Ca2+]i relationship was shifted to the left. These results suggest that changes in pHo can alter airway smooth muscle tone by changing [Ca2+]i and pHi.

Inhibitory effects of halothane on high(K+)-induced canine tracheal smooth muscle contraction and intracellular Ca2+ increment.

Halothane is a potent bronchodilator. The effects of halothane on isolated canine tracheal smooth muscle contraction and intracellular Ca(2+) increment induced by a high concentration of K+ were investigated to clarify how this anaesthetic decreases intracellular Ca2+ concentration ([Ca2+]i), an important second messenger. The tension of the muscle strips was measured using an isometric transducer, and [Ca2+]i was measured using a surface fluorescence spectroscopy. Exposure to a 72.7 mM K+ solution increased muscle tension and [Ca2+]i. Halothane (1,2,3 and 4% at the vaporiser) was introduced by bubbling in the presence of the 72.7 mM K+, and significantly decreased both this elevated muscle tension and the [Ca2+]i in a concentration-dependent manner. Similarly, slowly decreasing concentrations of K+ (48.5, 36.4, 24.2 and 18.2 mM) significantly decreased both of these variables. There was no significant difference between the slopes of the two regression lines of changes in muscle tension and changes in [Ca2+]i. From the evidence that tonic increase in [Ca2+]i by high concentrations of K+ is due to the influx of Ca2+ via L-type voltage channels, halothane may modify the L-type channels to decrease Ca2+ influx. In conclusion, halothane inhibits the high K(+)-induced canine airway smooth muscle contraction by decreasing [Ca2+]i. The decrease in [Ca2+]i by halothane might be due to inhibition of voltage-operated channels, especially the L-type channels.

Effect of nitric oxide on the colonic smooth muscle of patients with Hirschsprung's disease

Hirschsprung's disease results in bowel obstruction because of a failure of smooth muscle relaxation in both the aganglionic segment of bowel and the internal anal sphincter (IAS). Nonadrenergic noncholinergic (NANC) nerves, which use nitric oxide (NO) as their chemical messenger, are responsible for relaxing smooth muscle in normal bowel and the IAS. Previous work indicates that the cause of the aganglionic colon's inability to relax may be a lack of NANC nerves. To test this hypothesis, the authors compared the effect of an exogenous source of NO, S-nitroso-N-acetylpenicillamine (SNAP), on the isometric tension of smooth muscle strips taken from the ganglionic colon, aganglionic colon, and IAS of patients with Hirschsprung's disease. Exposure of ganglionic and aganglionic colon specimens to SNAP (10 −3 to 10 −5 mol/L) resulted in up to 70% reduction of resting tension. This relaxation occurred in a dose-dependent fashion and could be promptly reversed by the addition of the NO antagonist methylene blue. However, SNAP had no demonstrable effect on the smooth muscle strips taken from the IAS of patients with Hirschsprung's disease. This finding suggests that, in the aganglionic colon, a deficiency of NANC nerves contributes to the development of bowel obstruction. However, the failure of the IAS to relax in Hirschsprung's disease appears to be unrelated to NO and the NANC nervous system.

Nitric oxide modulates the calcitonin gene-related peptide-induced relaxation in guinea pig gallbladder strips in vitro.

Calcitonin gene-related peptide (CGRP) induces a dose-dependent relaxation in cholecystokinin-induced tension in guinea pig gallbladder strips in vitro. This effect seems to be modulated, in part, via activation of the nonadrenergic noncholinergic nervous system. Blockers of nitric oxide synthesis significantly reduced the CGRP effect. Increases in nitric oxide (NO) after treatment with CGRP suggested the involvement of NO in the observed relaxation.

Comparative studies on intracellular calcium and nadh fluorescence of the rabbit corpus cavernosum

Erectile function (erection and detumescence) involves the complex interaction of direct neuronal stimulation of corporal smooth muscle, neurohumoral release of specific endothelial contractile and relaxant factors, and secondary modulation by a variety of putative neuropeptides and vasoactive modulators. Using surface spectrofluorometry, we have correlated spontaneous contractile activity and the contractile response to field and pharmacological agents with intracellular calcium and NADH metabolism. The results demonstrate that the corpus cavernosal tissue has very unusual properties. Spontaneous contractile activity is correlated with a phasic increase in intracellular calcium. However, spontaneous contractile activity is most often correlated with a bi-phasic effect on the ratio of NADH/NAD. At the start of the spontaneous contraction, there is a sharp phasic increase in NADH/NAD; peak contractile force occurs simultaneous with a phasic decrease in this ratio showing that at peak force generation, there is a decrease in the level of intracellular energy. Phenylephrine stimulation results in an increase in intracellular calcium in proportion to the increase in tension; however, phenylephrine stimulation at low concentrations results in a net increase in the NADH/NAD ratio whereas high concentrations of phenylephrine result in a net decrease in the NADH/NAD ratio. In general, field stimulation results in a decrease in tension at low frequencies, a biphasic response at midfrequencies, and a contraction at high frequencies. These contractile responses are directly directly related to alterations in the intracellular concentration of calcium. That is, a decrease in tension is preceded by a decrease in intracellular calcium while an increase in tension is preceded by an increase in intracellular free calcium. Field stimulation results in a rapid and phasic alteration in the NADH/NAD ratio; however, the NADH/NAD response can be either an increase, decrease, or biphasic response. There does not appear to be a consistent relationship between the contractile/relaxant response to field stimulation and altered NADH/NAD ratio. Finally, ATP, bethanechol, and nitroprusside induce a decrease in the basal tension of the corpus cavernosal strips which corresponds with a decrease in the NADH/NAD ratio. However, whereas nitroprusside relaxation is correlated with a decreased intracellular calcium level, both ATP and bethanechol stimulate an increase in intracellular free calcium. These studies indicate that the response of the corpus cavernosal tissue to both field stimulation and pharmacological agents is complex and may involve both direct and indirect actions of a variety of cellular mediators on the corporal smooth muscle.

Pharmacological effect of ethanol on the function of rabbit corporal cavernosal tissue.

Pharmacologic and behavioral effects of alcohol on male sexual activity have long been controversial. Among the varied effects of alcohol, it has been described that ethanol reduces nitric oxide production. Appreciating the importance of nitric oxide-mediated regulation of corporal smooth muscle, this study was designed to investigate the in vitro pharmacologic effect of 1-5% ethanol on rabbit corpus cavernosum function. The results are summarized as follows. In isolated organ bath experiments, basal resting tension of corporal strips was significantly reduced with 3 and 5% ethanol exposure. Relaxation induced by field stimulation over a frequency range of 2-16 Hz was significantly decreased with 3 and 5% ethanol exposure. However, field-stimulated contraction at 32 Hz was reduced by 5% ethanol only. When corporal strips were preincubated with phenylephrine, 3 and 5% ethanol significantly inhibited field stimulated relaxation at all frequencies. Both phasic and tonic contraction induced by phenylephrine was significantly suppressed by 3 and 5% ethanol. KCl-induced contraction was decreased by 5% ethanol. ATP-induced relaxation was significantly enhanced by 1, 3 and 5% ethanol. Bethanechol-induced relaxation was significantly suppressed by 1, 3 and 5% ethanol. Direct nitroprusside-induced relaxation was not affected by any concentration of ethanol administration. These results demonstrated that ethanol had significant effects on both contraction and relaxation of rabbit corpus cavernosum. In general, corporal relaxation mediated through the acetylcholine-L-arginine-nitric oxide pathway was significantly more sensitive to ethanol than either ATP- or nitroprusside-induced relaxation.

Gamma-interferon modifies guinea pig airway functions in vitro

Cytokines produced by T-lymphocytes may have significant roles in the airway inflammation seen in bronchial asthma. Gamma interferon (IFN-gamma), a T-cell derived cytokine, is known to modify functions of both immune and non-immune cells. In this study, we investigated whether IFN-gamma can modify guinea pig airway functions in vitro. The isometric tension of guinea pig airway strips was measured in a tissue bath filled with Krebs-Henseleit solution. Contracting responses to carbachol and KCl, and the relaxing response to isoproterenol (ISO) were examined. Effects of IFN-gamma were examined by comparing responses of the strips incubated with or without IFN-gamma (1000 U.ml-1; 25,000 U.ml-1). Contracting responses to carbachol and KCl were not affected by the incubation with IFN-gamma other than slight increased in maximum contraction by carbachol after 5 hours incubation with 25,000 U.ml-1 of IFN-gamma. Both 1 and 5 h incubation of strips with 25,000 U.ml-1 IFN-gamma significantly increased the sensitivity to ISO (p < 0.01 and p < 0.05, respectively) without affecting maximum relaxation. The effect of IFN-gamma on ISO relaxation was abolished by the denudation of airway epithelium from strips, indomethacin (2 microM), and cycloheximide (70 microM) but not by N omega-nitro-L-arginine methyl ester (30 microM). In addition, heat-inactivated IFN-gamma and bacterial endotoxin (LPS, 0.625 pg.ml-1) had no effect on ISO relaxation. These results suggest that IFN-gamma is able to modify airway smooth muscle response to beta-adrenergic agonist by inducing release of prostanoids from airway epithelium.

Extracellular pH signals affect rat vascular tone by rapid transduction into intracellular pH changes.

1. Changes in extracellular pH (pHo) are known to produce large effects on vascular tone, but the mechanisms involved are not understood. As changes in intracellular pH (pHi) can also affect vascular tone, we have investigated the effects of changing pHo upon both pHi and tone. 2. Strips of rat mesenteric resistance vessels were loaded with the pH-sensitive fluorophore SNARF 1; thus tension and pHi could be simultaneously measured as pHo was altered. 3. Whenever pHo was altered there was a corresponding alteration of pHi. Furthermore, when pHo was changed the pHi change was more rapid than that reported to occur in other cells. The time to half-peak intracellular response was 38 +/- 4 s (n = 11). The induced pHi change was also less attenuated than in many other cells studied. Thus a ratio of 0.73 was obtained for the change in pHi per unit pHo change (n = 7). 4. An increase in pHi produced by an increase in pHo was accompanied by an increase in tension in the vascular strips. In other experiments pHi was increased at constant pHo by the addition of the weak base trimethylamine (40 mM). This also elevated tension in the strips. Conversely when pHo was changed while pHi was held at resting values, no change in vascular tone occurred. 5. It is concluded that the effects of pHo on vascular tone are due to the induced change in intracellular pH, and that the vascular smooth muscle cell is functionally well adapted to respond to changes in tissue pH, thereby allowing blood flow to a tissue to be rapidly altered to meet changing needs.

Role of calcium in mediating the biphasic contraction of the rabbit urinary bladder

1. 1. The response of the urinary bladder to field stimulation is biphasic in nature consisting of an initial phasic contraction followed by a prolonged tonic phase which lasts for the duration of the stimulation. 2. 2. The phasic response is mediated by the release of neurohumoral transmitters, primarily acetylcholine (via muscarinic receptor stimulation) and ATP (via purinergic receptor stimulation). The tonic component is mediated entirely via muscarinic receptor stimulation. 3. 3. The present study investigates the dependence on extracellular calcium of the phasic and tonic contractile responses to field stimulation, bethanechol, and ATP. The results can be summarized as follows: 4. 4. Field stimulation (2 and 32 Hz) and bethanechol evoke a biphasic contractile response whereas ATP evokes only a phasic response. 5. 5. There were no significant effects of either calcium channel blockers or calcium fee EGTA medium on either spontaneous contraction or basal tension of muscle strips. 6. 6. The calcium channel antagonists diltiazem and verapamil inhibited both the phasic and tonic responses induced by field stimulation (both 2 and 32 Hz) in a dose dependent manner. 7. 7. For both 2 and 32 Hz stimulation, the ED 50s for the inhibition of the tonic phases of the responses to field stimulation were significantly lower than the ED 50s for the inhibition of the phasic responses. 8. 8. The tonic phase of the responses to field stimulation were inhibited to a significantly greater degree than the phasic responses by incubation in calcium-free medium containing EGTA. 9. 9. Both the phasic and tonic components of the response to bethanechol stimulation were inhibited equally, and followed a similar time course as the tonic component of field stimulation. 10. 10. The response to ATP was relatively insensitive to calcium depletion. 11. 11. In summary, the tonic response to field stimulation (purely cholinergic) is significantly more dependent on extracellular calcium than is the phasic response (cholinergic + purinergic).

Mechanisms of caffeine-induced contraction and relaxation of rat aortic smooth muscle.

1. Using front-surface fluorimetry and Fura-2, we determined the effects of caffeine on cytosolic calcium concentration ([Ca2+]i) and on tension of strips of the rat thoracic aorta. We also determined the effects of caffeine on 45Ca2+ influx into the strips. The objective was to elucidate the mechanism of contraction and relaxation in vascular smooth muscle, as induced by caffeine. 2. In normal physiological salt solution (PSS), caffeine induced a transient tension development, while it induced a biphasic change in [Ca2+]i. The initial transient peak in [Ca2+]i which coincided with tension development was followed by a sustained increase. Thus, changes in tension did not follow changes in [Ca2+]i. In Ca(2+)-free PSS, both the caffeine-induced contraction and the increase in [Ca2+]i were transient. It was suggested that in both the presence and absence of extracellular Ca2+, the transient increase in [Ca2+]i was due to the release of Ca2+ from the intracellular store. Although the sustained increase in [Ca2+]i depended on extracellular Ca2+, it was not affected by diltiazem, a Ca2+ antagonist. 3. Caffeine inhibited the increase in [Ca2+]i and tension development during 118 mM-K+ depolarization, in a concentration-dependent manner. The extent of reduction in tension (relaxation) was greater than that expected from the reduction in [Ca2+]i based on the [Ca2+]i-tension relationship observed with K+ depolarization. Pretreatment of the strips with ryanodine did not alter the inhibitory effects of caffeine. 4. Caffeine inhibited the increased [Ca2+]i and developed tension during stimulation by 10(-5) M-noradrenaline, in a concentration-dependent manner. 5. Dibutyryl cAMP (10(-4) M) inhibited both high K(+)-induced and noradrenaline-induced tension development. Inhibition of an increase in [Ca2+]i in relation to the inhibition of tension during noradrenaline stimulation was much greater than that in 118 mM-K+ depolarization. 6. Although caffeine per se had no effect on 45Ca2+ influx in the strips in normal PSS, caffeine did inhibit the increase in 45Ca2+ influx stimulated by 118 mM-K+ or by 10(-5) M-noradrenaline, to a similar extent and with similar IC50 values. 7. The characteristic features of the effects of caffeine on vascular smooth muscle, i.e. the transient nature of contraction and the relaxation of precontracted strips could be explained as follows: caffeine is able to reduce [Ca2+]i after releasing Ca2+ from intracellular stores; however, this may play a minor role. Independent of the [Ca2+]i reduction, the second messenger, cAMP, might directly influence the [Ca2+]i-tension relationship, and if so, would play a major role.

Effects of prostanoids and indomethacin on isolated smooth muscle from the human lower oesophagus.

Smooth muscle strips representing the longitudinal and the circular muscle layers of the oesophageal body and the oesophagogastric junction of the human oesophagus were mounted in organ baths and isometric tension recorded. Transmural field stimulation was applied to strips from both layers of the oesophageal body and to strips from the longitudinal muscle layer of the oesophagogastric junction. This resulted in contractions after cessation of the stimulus train in circular muscle, and in contractions during stimulation in longitudinal muscle preparations. Indomethacin lowered active tension in strips from the circular muscle layer of the oesophagogastric junction, and decreased responses to transmural field stimulation in all strips from the longitudinal muscle layer. The response to transmural field stimulation was increased in strips from the circular muscle layer of the oesophageal body, suggesting an inhibitory function of endogenous prostanoids in this muscle layer. Prostaglandin (PG) F2 alpha and the thromboxane A2 mimic U-46619 increased the responses to transmural field stimulation in all muscle types and the active tension in circular muscle from the oesophagogastric junction. PGE1 and PGE2 increased the responses to transmural field stimulation strips from the longitudinal muscle layer. In circular muscle strips from the oesophageal body responses to transmural field stimulation were inhibited. PGE1 decreased active tension in circular muscle strips from the oesophagogastric junction, whereas PGE2 produced inconsistent effects. These results suggest a role of prostanoids in the regulation of neuromuscular function in the human lower oesophagus. Moreover, exogenous prostaglandins may modulate oesophageal motor function.

Effect of parathyroidectomy on serum levels of procollagen type I C-term extension peptide

Parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) have been shown to relax various types of smooth muscle, e.g. vascular, uterine and gastric. This study demonstrates that PTH and PTHrP both relaxed cholecystokinin octapeptide (CCK)-induced tension in guinea pig gallbladder strips. This relaxation was concentration-dependent. The use of PTHrP (7-34) blocked the relaxant effect of both agents. This suggested PTH and PTHrP were acting through the same receptor. The use of Rp-cAMPs, an inhibitor of cAMP activation of protein kinase A, and H-89, a selective inhibitor of protein kinase A, suggested that cAMP mediated the relaxant action of PTH and PTHrP. The use of iberiotoxin indicated that the high conductance Ca2+-activated potassium channels also mediated the actions of PTH/PTHrP. The use of KT5823, a selective blocker of protein kinase G, also decreased the amount of relaxation induced by PTH/PTHrP. This suggested that crosstalk between the two second messenger (cAMP and cGMP) systems occurred.

Bronchodilator action of an agonist for histamine H3-receptors in guinea pig perfused bronchioles and lung parenchymal strips.

Isolated guinea pig perfused bronchioles and lung parenchymal strips were examined as an in vitro model for assessment of the direct effect of pharmacologic agents on the airway smooth muscle. The experiments were performed with a perfusion technique in bronchioles, the input pressure being measured as an index of the state of dilation, while changes in tension of the lung parenchymal strips were measured with an isometric force transducer. In both preparations, histamine and acetylcholine elicited dose-related contractile responses whereas fenoterol induced a concentration-dependent relaxation. After the 3 agonists' activities were compared in these 2 preparations, we tested the intrinsic effects of a specific H3 agonist, (R) alpha-methylhistamine ([R] alpha-MeHA). Statistical analysis was by Student's t test on the Emax (expressed as a percentage of 10(-4) M papaverine relaxation), EC50, and slopes of regression lines calculated from the concentration-response curves plotted for (R) alpha-MeHA alone or in presence of antagonists. Our results showed that (R) alpha-MeHA induced a concentration-dependent relaxation of perfused bronchioles and lung parenchymal strips competitively inhibited by 10(-7) M thioperamide (H3-antagonist), whereas 10(-5) M cimetidine (H2-antagonist) failed to prevent this effect. These results suggest the presence of H3-histaminergic dilatory receptors in the guinea pig airway.

Basal and Acetylcholine-Stimulated Nitric Oxide Formation Mediates Relaxation of Rabbit Cavernous Smooth Muscle

Externally applied acetylcholine (ACh) in human corpus cavernosum has been shown to cause endothelium-dependent smooth muscle relaxation. Changes in isometric tension in rabbit cavernous smooth muscle strips mounted in organ bath chambers were monitored in the presence of blocking agents. Nitric oxide (NO) is known as an endothelium-derived relaxation factor (EDRF). Addition of specific inhibitors of nitric oxide synthesis, such as L-n-monomethyl arginine (L-NMMA) at 5 × 10−4 mol/l.. or L-n-nitro arginine (L-NOARG) at 2 × 10−4 mol/l. to strips precontracted with phenylephrine (PE) at 3.16 × 10−6 mol/l. led to significant increases in tension. In the presence of L-NMMA or L-NOARG, relaxing effects of ACh at 10−8−3.16 × 10−5 mol/l. mediated by muscarinic receptors were almost completely abolished.These data indicate that rabbit cavernous smooth muscle is under the control of basal NO release. They constitute strong evidence that cholinergically induced endothelial formation of NO plays a crucial role in relaxing cavernous smooth muscle.

Effects of pinacidil on contractile proteins in high K+‐treated intact, and in β‐escin‐treated skinned smooth muscle of the rabbit mesenteric artery

1. The effects of pinacidil were investigated on changes in cellular Ca2+ concentration ([Ca2+]i) and tension in intact and chemically skinned smooth muscle strips of the rabbit mesenteric artery. 2. High K+ (128 mM) produced a large phasic followed by a tonic increase in [Ca2+]i and tension in intact muscle strips. Pinacidil at 10 microM but not 1 microM, inhibited the phasic and tonic contractions induced by 128 mM K+ without a corresponding change in [Ca2+]i. 3. In beta-escin-treated skinned smooth muscle, the minimum Ca2+ concentration that produced contraction was 0.1 microM and the maximum contraction was obtained at 10 microM. Pinacidil at 10 microM but not 1 microM, shifted the pCa-tension relation curve to the right and also inhibited the maximum contraction induced by Ca2+. The concentrations of Ca2+ required for half maximal tension were 0.9 microM in control and 1.5 microM in the presence of 10 microM pinacidil. Calmodulin (2 microM) increased the contraction induced by 0.3 microM Ca2+ (but not by 10 microM Ca2+) in the skinned strips. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM or 10 microM Ca2+ in the presence of 2 microM calmodulin. 4. Noradrenaline (NA, 10 microM) with guanosine triphosphate (GTP, 3 microM), guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S, 3 microM) or 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.1 microM) all enhanced the contraction induced by 0.3 microM Ca2+. Pinacidil (10 microM) inhibited the contraction induced by 0.3 microM Ca2+ more strongly in the presence of the above agents than in their absence. 5. Following application of 2 mm adenosine-5'-O-(3-thiotriphosphate) (ATPyS) with 0.3 microM Ca2", 4mM MgATP produced contraction in skinned strips in Ca2 -free solution containing 4mM EGTA ('Ca2+- independent contraction'). The amplitude of the Ca2 +-independent contraction was almost the same as that obtained with 1O microM Ca2 . Pinacidil (1O microM) had no effect on the amplitude of the Ca2+-independent contraction nor did it have any effect on the contraction induced by a solution containing no MgATP ('rigor contraction'). 6. It is concluded that pinacidil (10 microM) acts directly on the contractile apparatus to inhibit Ca2'-induced contraction in smooth muscle of the rabbit mesenteric artery. The site of action of pinacidil may be between Ca2 +-calmodulin complex formation and phosphorylation of the myosin light chain.

Comparison of the effects of a bufodienolide and ouabain on neuronal and smooth muscle preparations

The effect of a bufodienolide (monohydroxy-14,15-epoxy-20,22-dienolide glycoside) purified from toad skin was compared with that of ouabain on 3H-noradrenaline release and on the tension of rabbit pulmonary arterial strips. This compound exerted an ouabain-like activity. The neuronal effects of this bufodienolide derivative on squid axon were also studied and compared with those of ouabain. Both compounds enhanced the resting and stimulation-evoked (2 Hz. 360 shocks) release of 3H-noradrenaline. Moreover, in the presence of either this bufodienolide or ouabain, the tension of the rabbit artery increased gradually, and the contraction evoked by electrical stimulation was potentiated. Both compounds enhanced, in a prazosin-sensitive way, smooth muscle responses to noradrenaline and to electrical stimulation. In higher concentrations, they contracted smooth muscle cells of pulmonary artery, an action which was insensitive to prazosin. The bufodienolide was about 8 times more active in inhibition of 22Na efflux than was ouabain, but did not affect Ca efflux, which is not sensitive to ouabain. It is therefore concluded that compounds with an inhibitory effect on Na +,K +-ATPase are able to affect chemical neurotransmission of blood vessels in such a way that in lower concentrations they potentiate the release of noradrenaline, and in higher concentrations they contract directly the smooth muscle. These findings indicate that such compounds if they are present in the circulation might be involved in the physiological regulation of blood pressure or in the genesis of hypertension.

The effect of aminophylline on function and intracellular pH of the rat diaphragm

We studied the effect of aminophylline on twitch tension (TT) and intracellular pH (pHi) in isolated rat diaphragm strips that were fatigued, hypercapnic, or hypoxic. Superfused muscles were directly stimulated at 0.5 Hz. The pHi was measured from distribution volumes of dimethyl-oxazolidinedione. Fatigue was induced by intermittent tetanic stimulation. Hypercapnia and hypoxia were produced by altering superfusate carbon dioxide tension (PCO2) or oxygen tension (PO2). Aminophylline (1.0 mmol.l-1) reversed the twitch decay seen during fatigue or hypercapnic acidosis, and caused partial recovery of twitch depression during hypoxia. Muscle fatigue was not due to an intracellular acidosis. Both hypercapnia and hypoxia lowered pHi. Aminophylline did not alter pHi in unstimulated muscles, but caused a significant fall in pHi in stimulated muscles that were fatigued or hypoxic. High dose aminophylline improved twitch tension in diaphragm strips that were fatigued, acidotic, or hypoxic. Twitch potentiation was not due to an intracellular alkalosis. Aminophylline lowered pHi in stimulated muscle, and thus, theoretically, could sometimes be harmful in the treatment of muscle fatigue.

Effect of aging on endothelium-dependent vascular relaxation of isolated human basilar artery to thrombin and bradykinin.

Using strips of human basilar arteries mounted in organ chambers to record isometric tension, we investigated vascular reactivity to thrombin and bradykinin. Both agents produced endothelium-dependent relaxation of basilar artery strips precontracted with phenylephrine but had no effect on resting tension in strips with or without endothelium. The relaxations caused by thrombin were abolished by antithrombin III/heparin, hirudin, and MD805. Thrombin but not bradykinin caused complete tachyphylaxis toward a second exposure. Indomethacin did not inhibit the relaxations induced by thrombin or bradykinin, whereas bromophenacyl bromide and methylene blue did. Aging decreased the relaxation induced by thrombin but did not affect the concentration needed to reach 50% maximal relaxation, nor did it affect the maximal relaxation in response to bradykinin, calcium ionophore A23187, and sodium nitroprusside. Our results suggest that thrombin and bradykinin produce endothelium-dependent relaxations mediated by an endothelium-derived relaxing substance and that the relaxation caused by thrombin is mediated by a proteolytic action on endothelial cells. The decrease in relaxations in response to thrombin with increasing age might be due to a decrease in the number or sensitivity of thrombin receptors on endothelial cells.

Atropine‐resistant relaxation induced by high K+ in iris dilator muscle of the rat and pig

1. The effects of high K+ ion concentration on the isometric tension in dilator muscle strips of the rat and porcine iris were examined. A high K+ solution, prepared by the replacement of Na+ in the medium with equimolar K+, was applied in the presence of 1 microM phentolamine, 1 microM propranolol and 1 or 10 microM atropine. High K+ (greater than 20 mM) induced a biphasic response; an initial phasic contraction followed by relaxation rather than tonic contraction. 2. An additional application of a Ca2+ antagonist, 1 microM nifedipine or nicardipine, almost completely blocked the K(+)-induced initial contraction and enhanced the following relaxation. The effect of K+ under these conditions was concentration-dependent in the range 20 to 80 mM. The maximum amplitude of the atropine-resistant relaxation induced by high K+ corresponds to 50-75% of that produced by acetylcholine in the absence of atropine. A similar K(+)-induced relaxation was observed in the porcine iris dilator. 3. The atropine-resistant relaxation in the rat iris dilator was not affected by pretreatment with 10 microM ouabain. The relaxation induced by 40 or 80 mM K+ in the porcine dilator was slightly enhanced or not affected, respectively, in the presence of 1 microM ouabain. Application of 10 microM ouabain per se induced relaxation in the porcine iris dilator. 4. The low Na+ ion concentration present in high K+ solutions was not responsible for the K(+)-induced relaxation since the complete replacement of Na in the medium with Tris did not affect significantly the relaxation produced by high K(+)-containing solutions. 5. Neither 1 microM tetrodotoxin, 10 microM indomethacin, 10 JM nordihydroguaiaretic acid nor hypoxic conditions affected the high K+-induced relaxation. 6. The inherent tone of the rat iris dilator was not affected by either 8-bromo cyclic GMP, dibutyryl cyclic GMP (0.1-0.3 mM) or nitroprusside (1-100 microM). 7. These results may suggest that the atropine-resistant relaxation induced by high K+ is not due to either activation of the Na-K pump or release of a relaxing factor produced by oxidative metabolism. Although the relaxation mechanism has not been elucidated, it is probably not mediated by an increase in cellular cyclic GMP levels.