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Isolation of a peptide material showing strong rectal muscle-contracting activity from chicken rectum and its identification as chicken neurotensin.

A peptide with strong activity to contract isolated whole chick rectum, RC-substance, in an acid-acetone extract prepared from 2.0 kg wet weight of chicken rectums was isolated by gel filtration on Sephadex G-25, ion exchange chromatography on a SP-Sephadex C-25 column, and high voltage paper chromatography. The close purity of this substance was established by high performance liquid chromatography (HPLC). The RC-substance displayed the same pharmacological and enzymatic properties as bovine neurotensin (NT). However, it differed from bovine NT in its behavior during the ion exchange chromatography, in its mobility during the electrophoresis, and in its retention time on HPLC. The amino acid analysis by a conventional OPA method revealed that all ten amino acid residues of which molar ratios were integral were common with those of chicken NT. Its biological activities were equipotent to those of bovine NT, suggesting the presence of proline in the COOH-terminal hexapeptide. From these results, it can be concluded that the RC-substance is identical to chicken NT. Exposure to high concentrations of bovine NT rendered the rectal smooth muscle of the chicken insensitive to subsequent applications of the RC-substance as well as bovine NT. This desensitization also reduced specifically the contractile responses of the rectal muscle elicited by non-adrenergic and non-cholinergic (NANC) nerve stimulation, suggesting a possible physiological role of the RC-substance as the neurotransmitter of NANC neurones.

Amino Acids↗

Increased permeability to sugar following muscle contraction. Inhibitors of protein synthesis prevent reversal of the increase in 3-methylglucose transport rate.

Contractile activity is followed by an increase in the permeability of muscle to sugar. The increase in sugar transport rate persists unchanged for hours before gradually returning to base-line (Holloszy, J. O., and Narahara, H. T. (1965) J. Biol. Chem. 240, 3493-3500). This prolonged delay suggested that the recovery process might involve protein synthesis. We have investigated this possibility. A large increase in permeability to [3-3H]methylglucose occurred in frog sartorius muscles stimulated to contract 60 times/min for 30 min. Cycloheximide or puromycin at concentrations that inhibited protein synthesis completely blocked reversal of the increase in permeability to 3-methylglucose. The recovery process was not irreversibly impaired, as permeability returned to base-line in muscles that were washed and incubated without protein synthesis inhibitors for a second 24-h period. Reversal of the increase in permeability to 3-methylglucose induced by a high concentration of epinephrine was also prevented by cycloheximide. In contrast, cycloheximide did not prevent reversal of the increases in sugar transport rate induced by either insulin or trypsin. These results provide evidence that exercise and catecholamines activate sugar transport by a process that requires protein synthesis for its reversal, while the increases in permeability induced by insulin and trypsin involve a different mechanism.

3-O-Methylglucose↗

Crossbridge behaviour during muscle contraction.

A number of recent observations by probe and X-ray methods on the behaviour of crossbridges during contraction is considered in relation to the energetics of the process. It is shown that a self-consistent picture of the crossbridge cycle, compatible with these observations and involving strongly and weakly attached crossbridges, can be obtained providing that the tension-generating part of the crossbridge stroke is only about 40 A i.e. about one-third of the usually accepted value. The myosin head subunits in the tension-generating bridges could have a configuration close to that of rigor. A mechanism is suggested whereby rapid tension recovery after quick releases up to 120 A could still be produced by such a system.

Actins↗

Regulation of the Ca2+ dependence of smooth muscle contraction.

Cellular mechanisms for the regulation of Ca(2+)-dependent myosin light chain phosphorylation were investigated in bovine tracheal smooth muscle. Increases in the free intracellular Ca2+ concentration ([Ca2+]i), light chain phosphorylation, and force were proportional to carbachol concentration. KCaM, the concentration of Ca2+/calmodulin required for half-maximal activation of myosin light chain kinase, also increased proportionally, presumably due to Ca(2+)-dependent phosphorylation of the kinase. Isoproterenol treatment inhibited agonist-induced contraction by decreasing [Ca2+]i and thereby light chain phosphorylation. Depolarization by increasing concentrations of KCl also resulted in proportional increases in [Ca2+]i, KCaM, light chain phosphorylation, and force. However, the [Ca2+]i required to obtain a given value of either light chain phosphorylation or KCaM was greater in KCl-depolarized tissues compared to carbachol-treated tissues. In muscles contracted with KCl, isoproterenol treatment resulted in diminished light chain phosphorylation and force without alterations in [Ca2+]i or KCaM. Thus, isoproterenol inhibition of KCl-induced contraction results from a cellular mechanism different from that found in agonist-induced contraction. In neither case does isoproterenol produce relaxation by altering the calmodulin activation properties of myosin light chain kinase.

Animals↗

Accelerated non-muscle contraction after subarachnoid hemorrhage: cerebrospinal fluid testing in a culture model.

The cause of chronic cerebral vasospasm after subarachnoid hemorrhage has been studied intensively, but it is still controversial whether the observable luminal narrowing should be attributed to the contraction of vascular smooth muscle cells or whether it results from some organic change in the wall. A proliferation of myointimal cells, accompanied by increased deposition of collagen, as well as myonecrosis, have been frequently observed several days after aneurysm rupture. Studies from our laboratory showed that these myointimal cells had characteristics identical to myofibroblasts. In this study, we quantitatively and morphologically examined the effect of cerebrospinal fluid on the ability of myofibroblasts to alter collagen matrices using an in vitro model. Myofibroblasts contract the collagen matrix by rearranging or compacting the framework of collagen fibers. Cerebrospinal fluid obtained from patients with recently ruptured aneurysms significantly accelerated lattice contraction, especially when the patient developed symptomatic vasospasm. This study suggests that myofibroblasts in the spastic artery can produce a contractile force that contributes to chronic vasospasm, tightening the proliferated collagen. Some unknown agent present in bloody cerebrospinal fluid accelerates the rearrangement of the collagen lattice by myofibroblasts, both of which have, until now, been considered non-contractile components.

Adult↗

[Parameters for determining the time of death from post-mortem muscle contraction--precision of assessing time of death].

The objectified muscular contraction on excitation with rectangular impulses of 1 sec duration and definite current intensity is in its postmortem course characterised by: 1. Change of the shape of muscular contraction (time-course of muscular contraction) from a two peak to a one peak shape 2. postmortem decrease of the maximum force of muscular contraction on definite current intensity 3. increase of the relaxation time of muscular contraction. On an random sample of fifty bodies decrease of the maximum force and increase of the relaxation time as criteria for extrapolating the time since death were investigated. The extrapolation was performed on the basis of the regression between ln of the maximum force and time using the mean values for slope and intercept and the regression between ln of the relaxation time and the ln of the time since death respectively using the mean values for slope and intercept too. On fifty bodies the calculated 95%--limits of confidence are +/- 2.85 h (decrease of the maximum force) and +/- 2.7 h (increase of relaxation time) up to 13 hpm. The calculated 95% limits of confidence were examined on an independent random sample of 20 bodies and proved to be reliable.

Electric Stimulation↗

Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction.

The effect of Ca2+ upon the rate constant of force redevelopment following a period of isotonic shortening with immediate restretch to the starting sarcomere length was studied in rabbit psoas fibers at 5 degrees C. Control experiments support the assumption that the rate constant of force redevelopment represents isometric cross-bridge turnover kinetics (fapp + gapp), where fapp and gapp are the rate constants characterizing the transitions from the non-force-generating states to the force-generating states and back to the non-force-generating states, respectively. Parallel measurements of the rate constant of force redevelopment and of force, stiffness, and fiber ATPase during isometric contraction allow the effect of Ca2+ upon fapp and gapp to be determined. Analysis reveals that Ca2+ has a marked effect upon fapp, while gapp remains approximately unchanged. Furthermore, in the range above 25-30% of maximum Ca2+ activation, regulation of force, stiffness, and ATPase is mediated through changes in fapp. Below this range, however, it cannot be ruled out that, in addition, cross-bridges are also switched in and out of the turnover process ("recruitment"). As a consequence of regulation through turnover kinetics, both Ca2+ sensitivity and the slope of force-pCa (-log[Ca2+]) relations are shown to be affected by the ratio fapp/gapp, which may represent an important mechanism of modulation of contractile function in addition to modulation through changes within the regulatory protein system.

Adenosine Triphosphatases↗

The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, inhibits porcine arterial smooth muscle contraction.

The Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) has been shown to suppress cytokine production in macrophages. We have examined the effect of OdDHL and related compounds on constrictor tone of porcine blood vessels. OdDHL (1-30 microM) caused a concentration-dependent inhibition of U46619-induced contractions of the coronary artery through a largely endothelium-independent mechanism, but was markedly less effective in the pulmonary artery. Quantitively similar effects to those produced by OdDHL were observed with N-(3-oxododecanoyl)-L-homocysteine thiolactone, a thiolactone derivative, while N-3-oxododecanamide, a lactone-free acyl analogue, possessed 1/3rd the potency as a vasorelaxant. Neither N-butanoyl-L-homoserine lactone nor L-homoserine lactone (up to 30 microM) were active. Our findings indicate that OdDHL inhibits vasoconstrictor tone of both pulmonary and coronary blood vessels from the pig. The vasorelaxant action of OdDHL appears to be primarily determined by the N-acyl chain length, with a minor contribution by the homoserine lactone moiety.

4-Butyrolactone↗

Relative contributions of extracellular Ca2+ and Ca2+ stores to smooth muscle contraction in arteries and arterioles of rat, guinea-pig, dog and rabbit.

1. These studies describe the functional effects of modulation of the sarcoplasmic reticulum (SR) Ca2+ stores at three levels of the vasculature: (i) large arteries (rat and guinea-pig aorta); (ii) small resistance arteries (rat tail artery, rabbit mesenteric artery, dog mesenteric artery); and (iii) arterioles (guinea-pig submucosal arterioles of the small intestine). 2. All tissues responded to phenylephrine (PE; 10 mumol/L) with a transient contraction in Ca(2+)-free Krebs', reflecting Ca2+ release from PE-sensitive Ca2+ stores. After pretreatment with cyclopiazonic acid (CPA; 30 mumol/L) or thapsigargin (TSG; 1 mumol/L), putative SR Ca2+ pump inhibitors, the PE-induced contraction in a Ca(2+)-free medium was significantly inhibited in arterial tissues at all levels of the vasculature. Similarly, ryanodine (RYA; 30 mumol/L), an agonist that enhances Ca2+ release from the SR, also reduced the PE contraction in a Ca(2+)-free solution. 3. CPA or TSG alone in the presence of extracellular Ca2+, caused marked and sustained contraction in the rat and guinea-pig aorta and marked but transient or no contraction in the resistance arteries. In the rat and guinea-pig aorta, RYA caused a slowly developing tension. Little increase in basal tension was produced by RYA in resistance arteries and arterioles. 4. The findings show that an agonist-releasable Ca2+ pool is present at all levels of the vasculature that is independent of the size of the vessels and suggest that under normal physiological conditions there is an intimate balance between the roles of the plasma membrane and of the SR in the maintenance of vascular contractility. It appears that the role of the SR diminishes as the arteries become smaller, while Ca2+ fluxes across the plasma membrane predominates.

Animals↗

Identification of a new vascular smooth muscle contracting polypeptide in Phoneutria nigriventer spider venom.

The fractionation of Phoneutria nigriventer spider venom by gel filtration (Sephadex G-10-120) followed by ion-exchange chromatography (microgranular CM-cellulose-52) resulted in sixteen fractions (CI to CXVI) from which CVII+VIII, CIX and CX+XI caused dose-dependent and short-lived contractions of both arterial and venous rabbit vessels. Fraction CX+XI was further purified by a reverse phase HPLC, and a contractile polypeptide (PNV2) was isolated. The amino terminal sequence of PNV2 (LAKRADICQPGKTSQRACET) indicated that it represents a pure polypeptide consisting of a single chain. Furthermore, the amino acid analysis of PNV2 revealed the presence of four disulfide bridges, a high content in Lys (14%), Glx (11%), and the absence of His. The global amino acid composition showed that this polypeptide is composed of 102 residues (Trp not included) with a calculated molecular weight of 12,114. Whether this peptide is responsible for the vascular alterations observed in Phoneutria envenomation, such as lung edema and priapism, remains to be further investigated.

Amino Acid Sequence↗

Selective M3 muscarinic receptor antagonists inhibit smooth muscle contraction in rabbit trachea without increasing the release of acetylcholine.

Although five muscarinic receptor subtypes have now been cloned, only three receptor subtypes have been demonstrated pharmacologically in bronchial tissue (designated M1, M2 and M3). By using drugs that show selectivity for these receptor subtypes, in combination with a sensitive and specific high-pressure liquid chromatography method that enables the direct measurement of acetylcholine (ACh) release, the existence and function of these muscarinic receptor subtypes was investigated in rabbit trachea in vitro. Atropine and ipratropium bromide, nonselective antimuscarinic agents, dose-dependently suppressed contraction of rabbit trachea induced by transmural electrical stimulation, but at the same time enhanced the release of ACh, suggesting the presence of an autoregulatory feed-back system. The M2/M4 receptor antagonists methoctramine, 11-((2-[(diethylamino)methyl]-1-piperidinyl)acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one, 5,11-dihydro-11-([(2-(2-[(dipropylamino)methyl]-1-piperidinyl)ethyl) amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one and 11-((4-[4-(diethylamino)butyl]-1-piperidinyl)acetyl)-5, 11-dihydro-6H-pyrido(2,3-b)(1,4)benzodiazepine-6-one also dose-dependently increased ACh release during electrical stimulation, indicating that the powerful negative feedback system is mediated by presynaptic autoreceptors of the M2 or M4 type.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

Neuromuscular blocking effects of aminoglycoside antibiotics on fast- and slow-contracting muscles of the cat.

The neuromuscular-blocking effects of neomycin, streptomycin, and gentamicin were examined in fast- and slow-twitch muscles of pentobarbital-anesthetized cats. These antibiotics were more effective in reducing indirectly stimulated contractions of the gastrocnemius muscle than of the ipsilateral soleus muscle in adult cats. Respiratory-depressant effects of these antibiotics were more closely associated temporally with effects on soleus than on gastrocnemius twitch. Neomycin was a more potent neuromuscular blocking agent in kittens greater than 7 weeks old than in kittens less than 6 weeks old. Calcium ions were more effective in antagonizing the effects of neomycin on gastrocnemius than on soleus twitch; however, neostigmine was more effective in antagonizing the effects of neomycin on soleus than on gastrocnemius twitch. These data indicate a difference in responsiveness of fast- and slow-twitch muscles (fast greater than slow) to the neuromuscular-blocking effects of aminoglycoside antibiotics, and suggest that this may be related to a difference in the predominance of pre- and postjunctional inhibitory actions of these agents in the 2 muscle types.

Aminoglycosides↗

An automated superfusion technique for measuring circular muscle contraction. Effects of serotonin on intra- and extracranial arteries.

A new in vitro technique is described and its advantages are demonstrated: "true" circular contraction is measured; arteries are in cascade, permitting comparison of intra- and extracranial arteries from the same animal; the mechanical influences on contractility are reduced; long-lasting experiments of up to 50 h can be performed; the procedure is fully automated. The maximal effect of an agonist was not only markedly modified during the first 2--4 h after fixing the arteries in the apparatus but also slightly during the next 3--4 h. After the initial stabilization period, the arterial response to an agonist remained highly reproducible for 24--48 h (standard deviation not exceeding 7%). This was demonstrated in all the types of arteries tested. With serotonin as agonist, there was a significant difference in --log ED50 values for intracranial arteries (basilar 8.70, middle cerebral 8.72) vs. extracranial (lateral nasal 8.15; facial 8.14) and peripheral (branch of saphenous 8.14) arteries.

Animals↗

Effects of tetrandrine on smooth muscle contraction induced by mediators in pulmonary hypertension.

AIM: In attempt to characterize tetrandrine on pulmonary hypertension, biological activities induced by a range of mediators implicated in the pathogenesis of pulmonary hypertension were investigated. METHODS: Pulmonary artery rings and tracheal segments were contracted with couples of bioactive substances in which a series experiments including effects of tetrandrine on calcium agonist, endothelin, thromboxane A2, angiotensin II, neuropeptide Y, histamine, 5-methyl furmethide were performed, the influences of tetrandrine in the concentration of 1 to 30 micromol/L were investigated. RESULTS: Tetrandrine inhibited calcium agonist BayK8644, endothelin-1 and thromboxane A2 mimetic U46619, angiotensin II- and neuropeptide Y-induced contractile responses with depression of the maximal contraction of pulmonary artery rings in a varying extent. Tetrandrine inhibited leukotriene E4-induced concentration-response curve in a competitive antagonist manner with a pKB of (5.29+/-0.11) without any influence leukotriene C4, leukotriene D4, histamine, and 5-methyl furmethide induced contractile responses of guinea pig trachea. CONCLUSION: Tetrandrine may produce multiple pharmacological effects against calcium channel antagonist, U46619, endothelin-1,angiotension II, and neuropeptide Y induced vasoconstriction in rat pulmonary arteries in varying extent and inhibition of leukotriene E4 rather than C4, D4, histamine, and 5-methyl furmethide induced contractile responses on rat tracheal segments. These pharmacological characteristics are considered to contribute to its antihypertensive action during pulmonary hypertension.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

Relationship between perineal muscle contractions, penile tumescence, and penile rigidity during nocturnal erections.

The mechanism of penile erection and erectile dysfunction is still unclear and widely debated. The role of the perineal muscles in the erectile process, especially in changes in intracavernous pressure, is increasingly being studied on the hypothesis that perineal muscular contractions are essential to full penile rigidity. In a previous investigation we studied the correlation between voluntary perineal muscle contractions and intracavernous pressure during artificially induced erections. The purpose of the current study was to examine whether under normal conditions of nocturnal erection a similar relationship exists between the electromyographic activities of perineal muscles and changes in penile rigidity. Nocturnal penile recordings were made of seven volunteers with psychogenic erectile dysfunctions. During nocturnal erections simultaneous computerized recordings were made of penile tumescence, penile rigidity, and electromyographic activities of perineal muscles. The peaks for the three variables were reached simultaneously. The results of this study suggest the existence of two different physiologic phases: a vascular phase and a muscular phase. Furthermore, in cases of dysfunctions, specific diagnostic assessment and therapeutic management will be required for both penile tumescence and penile rigidity.

Adult↗

Interneurone bursts are spontaneously associated with muscle contractions only during early phases of mouse spinal network development: a study in organotypic cultures.

For a short time during development immature circuits in the spinal cord and other parts of the central nervous system spontaneously generate synchronous patterns of rhythmic activity. In the case of the spinal cord, it is still unclear how strongly synchronized bursts generated by interneurones are associated with motoneurone firing and whether the progressive decline in spontaneous bursting during circuit maturation proceeds in parallel for motoneurone and interneurone networks. We used organotypic cocultures of spinal cord and skeletal muscle in order to investigate the ontogenic evolution of endogenous spinal network activity associated with the generation of coordinate muscle fibre contractions. A combination of multiunit electrophysiological recordings, videomicroscopy and optical flow computation allowed us to measure the correlation between interneurone firing and motoneurone outputs after 1, 2 and 3 weeks of in vitro development. We found that, in spinal organotypic slices, there is a developmental switch of spontaneous activity from stable bursting to random patterns after the first week in culture. Conversely, bursting recorded in the presence of strychnine and bicuculline became increasingly regular with time in vitro. The time course of spontaneous activity maturation in organotypic slices is similar to that previously reported for the spinal cord developing in utero. We also demonstrated that spontaneous bursts of interneurone action potentials strongly correlate with muscular contractions only during the first week in vitro and that this is due to the activation of motoneurones via AMPA-type glutamate receptors. These results indicate the occurrence in vitro of motor network development regulating bursting inputs from interneurones to motoneurones.

Action Potentials↗

Mechanism of smooth muscle contraction and relaxation mediated by kinin receptor.

The increase in sensitivity of guinea pig preparations to bradykinin (BK) due to stretching occurring with time after mounting was studied by determining the time course of changes in the cell membrane potential, measured with intracellular microelectrodes. A sustained hyperpolarizing effect of BK, which was observed in recently mounted preparations, became transient after 120 min, when it was followed by depolarization, which was much more evident after 4 h of stretching. As a consequence, a parallel increase in the contractile response to BK was also observed. The hyperpolarizing effect was due to the opening of Ca(2+)-dependent K+ channels sensitive to apamin, since BK dose-response curves done within 1 h of mounting were shifted to the left, becoming similar to dose-response curves obtained 4 h after mounting of the guinea pig ileum preparation. These results were specific for BK, since the potentiating effect of apamin was not observed for acetylcholine. Our results show that the activation of B2 receptors by BK in the isolated guinea pig ileum induce a dual effect--hyperpolarization and depolarization--and that the increase in the contractile response consequent to stretching is probably due to the inactivation of apamin-sensitive Ca(2+)-dependent K+ channels.

Animals↗

A calponin peptide enhances Ca2+ sensitivity of smooth muscle contraction without affecting myosin light chain phosphorylation.

In permeabilized smooth muscle, exogenously applied calponin binds to myofibrils and reduces Ca(2+)-activated tension (Itoh, T., Suzuki, S., Suzuki, A., Nakamura, F., Naka, M., and Tanaka, T. (1994) Pflügers Arch. Eur. J. Physiol. 427, 301-308). A calponin peptide (calponin Phe173-Arg185), which inhibits the binding of calponin to actin, blocks the action of calponin and enhances the contraction induced by submaximal Ca2+ in permeabilized vascular smooth muscle. Unlike calmodulin, this peptide enhances the Ca(2+)-induced contraction without a corresponding increase in the level of myosin light chain phosphorylation. These results suggest that calponin decreases the sensitivity of smooth muscle to Ca2+ at a given level of myosin light chain phosphorylation.

Actins↗