Kinetic analysis of contraction-relaxation process in smooth muscles

A method is proposed for analysing kinetic curves of the smooth muscle contraction--relaxation approved on smooth muscle preparations of ureter and taenia coli. A notion of the velocity coefficient is introduced. It is the parameter which gives a quantitative characteristics of changes in the dynamics of muscle contraction and relaxation under the effect of factors modifying the contractile response. In order to illustrate the application of this parameter studies were carried out of the effect of sodium-free medium, monensin, and of temperature decrease on the contractile activity of the smooth muscle tissue of the ureter. The method can be useful while investigating the regularities of electro- and pharmacomechanical conjunction in the smooth muscles, as well as during pharmacological screening of compounds--regulators of calcium homeostasis in myocytes and their contractile activity.     

A journey from benzanilides to dithiobenzanilides: Synthesis of selective spasmolytic compounds

A series of dithiobenzanilide derivatives was synthesized and each compound was evaluated for its ability to reduce KCl-induced contractions of smooth muscle preparations of the guinea pig. Starting from a recent publication describing benzanilide derivatives as antispasmodic agents, structure-activity guided synthesis was performed to obtain compounds with improved spasmolytic activity. First, compounds with two amide bonds were designed and second, both amide oxygens were replaced by two sp² sulfur atoms resulting in dithiobenzanilide derivatives. The most potent antispasmodic dithiobenzanilide 19 showed improved activity with an IC₅₀ value of 0.4 ??M. Moreover, the study also demonstrated that these active compounds were able to antagonize the effect of spasmogens like acetylcholine and phenylephrine and that the activity is not mediated by activation of ATP-dependent potassium channels (K_ATP-channels) or inhibition of endothelial nitric oxide synthase (eNOS).     

Spasmolytic flavonoids from Syzygium samarangense (Blume) Merr. & L.M. Perry

The hexane extract of Syzygium samarangense (Ss.Hex) dose-dependently (10-1000 microg/ ml) relaxed the spontaneously contracting isolated rabbit jejunum. Four rare C-methylated flavonoids with a chalcone and a flavanone skeleton were isolated from Ss.Hex and were subsequently tested for spasmolytic activity. All flavonoids, identified as 2'-hydroxy-4',6'-dimethoxy-3'-methylchalcone (1), 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (2), 2',4'-dihydroxy-6'-methoxy-3'-methylchalcone (3), and 7-hydroxy-5-methoxy-6,8-dimethyl-flavanone (4), showed dose-dependent spasmolytic activity in the rabbit jejunum with IC50 values of 148.3 +/- 69.4, 77.2 +/- 43.5, 142.4 +/- 58.6 and 178.5 +/- 37.5 microg/ml (mean +/- SEM), respectively. The dihydrochalcone derivative of compound 1, 2'-hydroxy-4',6'-dimethoxy-3'-methyldihydrochalcone (5), when tested for spasmolytic activity, did not significantly relax the smooth muscle relative to the other compounds. Verapamil, a standard spasmolytic, has an IC50 value of 0.16 +/- 0.04 microg/ml. This is the first report of the relaxant activity of chalcones, specifically of compounds 1-3.     

In-vitro characterization of the pharmacological effects induced by (-)-α-bisabolol in rat smooth muscle preparations

The present study deals with the pharmacological effects of the sesquiterpene alcohol (-)-α-bisabolol on various smooth-muscle preparations from rats. Under resting tonus, (-)-α-bisabolol (30-300?μmol/L) relaxed duodenal strips, whereas it showed biphasic effects in other preparations, contracting endothelium-intact aortic rings and urinary bladder strips, and relaxing these tissues at higher concentrations (600-1000?μmol/L). In preparations precontracted either electromechanically (by 60?mmol/L K(+)) or pharmacomechanically (by phenylephrine or carbachol), (-)-α-bisabolol showed only relaxing properties. The pharmacological potency of (-)-α-bisabolol was variable, being higher in mesenteric vessels, whereas it exerted relaxing activity with a lesser potency on tracheal or colonic tissues. In tissues possessing spontaneous activity, (-)-α-bisabolol completely decreased spontaneous contractions in duodenum, whereas it increased their amplitude in urinary bladder tissue. Administered in vivo, (-)-α-bisabolol attenuated the increased responses of carbachol in tracheal rings of ovalbumin-sensitized rats challenged with ovalbumin, but was without effect in the decreased responsiveness of urinary bladder strips in mice treated with ifosfamide. In summary, (-)-α-bisabolol is biologically active in smooth muscle. In some tissues, (-)-α-bisabolol preferentially relaxed contractions induced electromechanically, especially in tracheal smooth muscle. The findings from tracheal rings reveal that (-)-α-bisabolol may be an inhibitor of voltage-dependent Ca(2+) channels.     

Pharmacological actions of isoprostane metabolites and phytoprostanes in human and bovine pulmonary smooth muscles

We examined the responses to various isoprostane derivatives in bovine/human airway and pulmonary arteries. All biological activity of 15-F(2t)-IsoP was lost in its two major metabolites (15-keto-15-F(2t)-IsoP and 13,14-dihydro-15-keto-15-F(2t)-IsoP). We also examined the effects of several metabolites of 15-F(2t)-IsoP synthesized within our own laboratory-both epimers of 2,3-dinor-15-F(2t)-IsoP and of 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP, as well as 20-carboxy-2,3,4,5-tetranor-15 oxo-5,6,13,14-tetrahydro-15-F(2t)-isoP)-finding none of these to have any substantial excitatory effect. Finally, several plant-derived isoprostanes ("phytoprostanes") synthesized within our laboratory elicited little or no excitatory response in these three pulmonary smooth muscle preparations. We conclude that, although isoprostane exhibit powerful constrictor effects on airway and pulmonary vascular smooth muscles, metabolic processing of those isoprostanes essentially abolishes those biological actions; also, the phytoprostanes lack any appreciable pharmacological activity on those smooth muscle preparations.     

Purification and characterization of bioactive peptides from skin extracts of Rana esculenta

The peptide fraction extracted by methanol from the skin of Rana esculenta, a species widely distributed in Western Europe, was investigated. The pharmacological activity found in the extract is attributable to the presence of authentic bradykinin, together with a shorter, partially active version of this molecule, des-Arg9-bradykinin. Also the bradykinin fragment 1-7 has been isolated, but it was inactive in our bioassay system. Moreover, a family of hydrophobic peptides has been purified and characterized, which appeared devoid of pharmacological activities when tested on smooth muscle preparations, but were provided with hemolytic activities.     

Synthesis and contractile activity of new acetylenic and allenic analogues of leukotrienes C4 and D4: importance of the Z-11,12 double bond

The (5S,6R) isomers of new acetylenic and allenic analogues of leukotrienes C4 and D4 were synthesized for comparative pharmacological studies on intestinal smooth muscle preparations. These new analogues are poor spasmogenic agonists, the replacement of the 11,12-ene with a relatively more stable triple bond causing an important reduction in intrinsic activity. They did not show any significant antagonist activity. Unexpectedly, these results prove that the 11,12 portion in the triene structure of the lipophilic chain is critical for an agonist activity.     

Spasmolytic and tonic effect of Iberogast® (STW 5) in intestinal smooth muscle

STW 5 (Iberogast^®) is a fixed combination of nine medicinal plant extracts effective in the treatment of functional dyspepsia and irritable bowel syndrome. The effects of STW 5, a combination of Iberis amara fresh plant extract, and other eight plant extracts as well as single extract components including extracts from Menthae piperitae folium, Matricariae flos and Liquiritiae radix, were assayed in guinea pig ileum with or without stimulation with acetylcholine or histamine, in order to find a possible effect on the contractility of intestinal smooth muscle.STW 5 decreased acetylcholine- and histamine-induced contraction of guinea pig ileum. This was also true for extracts of Menthae piperitae folium, Matricariae flos and L. radix. Extract from I. amara, however, showed no spasmolytic action; in contrary, it increased the basal resting tone and contraction of atonic ileal segments. This was also true when STW 5 was employed.A spasmolytic action of STW 5 could also be observed in duodenum, jejunum and colon.These data are the first to show not only the spasmolytic effects of STW 5 and its component extracts in intestinal muscle but also the tonicising effects of STW 5 through its component Iberis amara extract in relaxed intestinal muscle. Thus, pharmacological evidence suggests a dual-action principle and may explain, at least in part, the clinically observed therapeutic efficacy of STW 5 (Iberogast^®) in both hypotonic and spastic dysmotility symptoms of functional dyspepsia and irritable bowel syndrome.     

Photoaffinity labeling of P2-purinergic and H1-histamine receptors in intact smooth muscle

This paper discusses the general applicability of photoaffinity labels as pharmacological receptor antagonists in functional studies of intact smooth muscle preparations. Guidelines are suggested that take into account the criteria for photoaffinity labeling studies as well as those for use with conventional antagonists.     

Bladder Smooth Muscle Strip Contractility as a Method to Evaluate Lower Urinary Tract Pharmacology

We describe an in vitro method to measure bladder smooth muscle contractility, and its use for investigating physiological and pharmacological properties of the smooth muscle as well as changes induced by pathology. This method provides critical information for understanding bladder function while overcoming major methodological difficulties encountered in in vivo experiments, such as surgical and pharmacological manipulations that affect stability and survival of the preparations, the use of human tissue, and/or the use of expensive chemicals. It also provides a way to investigate the properties of each bladder component (i.e. smooth muscle, mucosa, nerves) in healthy and pathological conditions.The urinary bladder is removed from an anesthetized animal, placed in Krebs solution and cut into strips. Strips are placed into a chamber filled with warm Krebs solution. One end is attached to an isometric tension transducer to measure contraction force, the other end is attached to a fixed rod. Tissue is stimulated by directly adding compounds to the bath or by electric field stimulation electrodes that activate nerves, similar to triggering bladder contractions in vivo. We demonstrate the use of this method to evaluate spontaneous smooth muscle contractility during development and after an experimental spinal cord injury, the nature of neurotransmission (transmitters and receptors involved), factors involved in modulation of smooth muscle activity, the role of individual bladder components, and species and organ differences in response to pharmacological agents. Additionally, it could be used for investigating intracellular pathways involved in contraction and/or relaxation of the smooth muscle, drug structure-activity relationships and evaluation of transmitter release.The in vitro smooth muscle contractility method has been used extensively for over 50 years, and has provided data that significantly contributed to our understanding of bladder function as well as to pharmaceutical development of compounds currently used clinically for bladder management.     

Effects of sodium nitroprusside, nitroglycerin, and sodium azide on levels of cyclic nucleotides and mechanical activity of various tissues.

Three agents that activate guanylate cyclase, sodium nitroprusside, nitroglycerin and sodium axide, were examined for their effects on cyclic GMP and cyclic AMP accumulation and muscle motility with several tissues. All of these agents, except nitroglycerin with ventricle preparations, increased cyclic GMP levels and did not alter cyclic AMP in incubations of preparations of bovine tracheal smooth muscle, guinea pig tracheal chains, taenia cecum, atria and ventricle, and rat liver and cerebral cortex. Increases in cyclic GMP with these agents occurred with relaxation of smooth muscle preparations and without alteration in the contractility of atrial preparations. These observations support the hypothesis that cyclic GMP accumulation in smooth muscle may be related to relaxation rather than contraction as proposed previously. Relaxation with these agents is not associated with alterations in cyclic AMP levels. Increases in cyclic GMP levels in atrial preparations can also occur without changes in contractile force or rate of contraction.     

The long-term influence of decentralisation or preganglionic hypogastric nerve stimulation in vivo on the reinnervation of minced vas deferens in the guinea-pig

Previous studies have shown that minced regenerating smooth muscle of the guinea-pig vas deferens becomes reinnervated by nerves growing in from the surrounding intact vas deferens. Using electron microscopy, we have examined the effect of altering activity in the preganglionic nerves, either by decentralisation, or by chronic stimulation of the hypogastric nerve, in vivo, on the reinnervation of regenerating smooth muscle cells. Chronic stimulation induced earlier reinnervation than that seen in unstimulated (sham-operated) or decentralised preparations; the number of nerve profiles present in four preparations stimulated for up to 7 days was approximately 10-20 times that seen in unstimulated or decentralised preparations. However, electron micrographs revealed that "empty" nerve terminals were a feature following stimulation for longer periods. Decentralised preparations showed little change of reinnervation, at least up to 7 weeks. Compensatory changes in the density of innervation were found in the unstimulated contralateral vas deferens.     

Biochemical basis for the spasmolytic effect of isoquinoline and phenothiazine derivatives

Experiments with isolated ring-like strips of pig heart veins and arteries and those with rat small intestinal strips made in the presence of hyperpotassium contracture have demonstrated that spasmolytic activity of papaverine, nospa, nonachlazine, and ethmozine is a consequence of the reduced respiratory intensity and phosphorylation in smooth muscle mitochondria. No changes in the anaerobic glycolysis rate have been found after drug administrations.     

Effect of papaverine on the longitudinal and circular smooth muscle of the guinea-pig caecum.

Electrical and mechanical activity of smooth muscle of the guinea-pig caecum was recorded by means of the sucrose-gap technique. The responses of longitudinal and circular smooth muscle to acetycholine were differently affected by changes of extracellular calcium concentration (0.8, 2.5 and 7.5 mM). The contractions of both preparations were depressed at high Ca++ concentrations, whereas at low Ca++ concentrations only contractions of the circular smooth muscle were augmented. The stimulatory effect of acetylcholine was decreased by papaverine in both preparations at all three concentrations of Ca++. This inhibition was the greater the lower the concentration of extracellular Ca++ and this process was more pronounced in the circular muscle. The ability of papaverine to counteract the effect of lowered concentrations of extracellular Ca++ on membrane excitability may well explain its inhibitory effect upon intestinal smooth muscle.     

Phenolic compounds from Gastrodia rhizome and relaxant effects of related compounds on isolated smooth muscle preparation

Gastrol (1), together with 10 known phenolic compounds, has been isolated from the MeOH extract of the rhizomes of Gastrodia elata Blume (Orchidaceae), and their structures were elucidated by detailed spectral analyses including by 2D NMR spectroscopic analyses. The relaxant effects of these constituents on smooth muscle preparations isolated from guinea-pig ileum were also studied in order to reveal their characteristic pharmacological activities.     

Parallel bioassay of 27 bombesin-like peptides on 9 smooth muscle preparations. Structure-activity relationships and bombesin receptor subtypes

Thirteen natural bombesin-like peptides and 14 synthetic analogues were submitted to parallel bioassay on 9 smooth muscle preparations in order to determine their relative potency, in comparison to bombesin and litorin. The natural peptides of the bombesin subfamily showed a uniformly high or moderate potency on all preparations. However, synthetic bombesins of shorter chain length (hepta- and octapeptides) manifested a good potency only on the rat uterus preparation. Among the peptides of the litorin and phyllolitorin subfamilies, only litorin and ranatensin presented a full spectrum of potency, equalling or even surpassing that of bombesin. All other natural and synthetic members of the two subfamilies showed a sharply dissociated spectrum of potency on the different smooth muscle preparations. The only exception was the rat urinary bladder and, in part, the chicken intestine, on which the peptides displayed a uniformly high potency, comparable to, or even greater than that of bombesin. The present results help to explain structure/activity relationships and suggest the probable existence, in the periphery, of multiple bombesin receptor subtypes.     

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.     

Diminished nitroprusside-induced relaxation of inflamed colonic smooth muscle in mice.

The dextran sodium sulphate (DSS) induced colitis in mice was used as a experimental model to study the contractility of murine longitudinal colonic smooth muscle during inflammation. Smooth muscle segments of proximal, middle and distal colon were mounted in organ baths. Smooth muscle contraction was induced by carbachol showing an aboral increase in activity, whereas in the inflamed middle colonic segment a marked decrease in activity was observed. The dilatative effect of sodium-nitroprusside (SNP) as a nitric oxide donor was investigated after precontraction by carbachol. Both in normal and DSS segments administration of SNP to isolated mouse colonic smooth muscle preparations caused regional differences in relaxation, the highest relaxation seen in normal proximal colonic tissue. However, this relaxation was markedly reduced in inflamed proximal preparations, associated with a diminished cGMP contents.