Evidence for selective sulfhydryl reactivity in cytochalasin A--mediated bacterial inhibitions.

Cytochalasin A (CA) at 5 × 10^?5$\text{M}$ strongly inhibits glucose transport in Arthrobacter sialophilis. This effect and other bacteriostatic and metabolic inhibitions of gram-positive bacteria are not caused by the closely related congeners cytochalasin B or D. Inhibitions by CA are nullified by prior drug incubation with sulfhydryl compounds. It was also found that the characterized adduct of CA with β-mercaptoethanol is devoid of biological activity. N-ethylmaleimide, p-chloromercuribenzoate and ethacrynic acid (a known, liposoluble, sulfhydryl reactant) were each shown at 5 × 10^?5$\text{M}$ to be relatively ineffective in inhibiting D-glucose transport in $\text{A. sialophilus}$. These observations suggest that CA reacts at the molecular biological level in a site-specific manner.     

A Possible role of the nucleus in cytochalasin B-Induced capping

The possible influence of the nucleus on Cytochalasin B (CB)-induced capping of antibodies to surface antigens on L cells SV40-3T3 and NRK La 334 cells was studied. The cap formation induced by CB, was generally localized opposite the nucleus which was displaced against the cell periphery. To be able to observe the nuclear membrane in relation to the capping process we have taken advantage of an antiserum specific for antigens in the nuclear membrane but lacking reactivity to the plasma membrane and intranuclear antigens. This approach indicated that the CB-induced capping caused an accumulation of nuclear membrane antigens in the area of the nucleus facing the cap. The CB-induced local accumulation of nuclear membrane antigens required intact cells and could not be induced by binding of antibodies to the nuclear membrane followed by exposure to CB. Whatever the basis for the CB-induced altered reactivity of the anti-nuclear membrane antibodies (folding of the nuclear periphery, for example) this result indicated that the nuclear membrane was affected by CB capping. The possible role of the nucleus in the CB-induced capping process was further investigated in enucleated cells. The results obtained indicate that such cells both when enucleated in suspension and adherent to a surface did not exhibit CB capping. This disappearance of CB capping did probably not reflect decreased cell viability, previous exposure of the cells to CB during the enucleation procedure or a decreased capacity of the enucleated cells to bind CB.     

The role of calcium at the sarcolemma in the control of myocardial contractility

The importance of sarcolemmal-bound calcium (Ca) in the control of contraction in mammalian myocardium is indicated by the following results. The curve that relates [Ca]o (from 50 microM to 10 mM) to force development and that which relates [Ca]o to Ca bound to a highly purified sarcolemmal fraction are superimposable. The ability of a series of cations to uncouple excitation from contraction is the same as their relative ability to displace Ca from the sarcolemma. Dimethonium, which specifically displaces cation from the diffuse double layer of the cellular surface, has little effect on contractile force. This indicates that the Ca actually bound to the sarcolemma is the surface Ca important in contractile control. Polymyxin B, a highly charged cationic amphiphilic peptidolipid, specifically competes for Ca-binding sites on anionic and zwitterionic phospholipid. It is a potent displacer of Ca from myocardial cells and purified sarcolemma and a potent uncoupler. Phospholipase D cleaves the nitrogenous base from sarcolemmal phospholipid with production of anionic phosphatidic acid. Phospholipase D treatment increases Ca bound to cells and purified sarcolemma and increases force development of ventricular tissue from both neonatal rat and adult rabbit. Insertion of charged amphiphiles in the sarcolemma as phospholipid analogues modulate interaction of Ca with the sarcolemma, e.g., anionic dodecylsulfate increases Ca bound to sarcolemmal vesicles by more than 80% and increases force development in rabbit papillary muscle by 100%. The effect of pH variation on Ca binding to phospholipid extracted from sarcolemma indicates that phospholipid accounts for at least 75% of the binding. The current model proposes a two-site control of Ca binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of voltage-gated potassium channels in the regulation of mouse uterine contractility

Background  

Uterine smooth muscle cells exhibit ionic currents that appear to be important in the control of uterine contractility, but how these currents might produce the changes in contractile activity seen in pregnant myometrium has not been established. There are conflicting reports concerning the role of voltage-gated potassium (Kv) channels and large-conductance, calcium-activated potassium (BK) channels in the regulation of uterine contractility. In this study we provide molecular and functional evidence for a role for Kv channels in the regulation of spontaneous contractile activity in mouse myometrium, and also demonstrate a change in Kv channel regulation of contractility in pregnant mouse myometrium.     

The role of STIM1 and SOCE in smooth muscle contractility

Contraction is a central feature for skeletal, cardiac and smooth muscle; this unique feature is largely dependent on calcium (Ca²⁺) signaling and therefore maintenance of internal Ca²⁺ stores. Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane protein that functions as a Ca²⁺ sensor for the activation store-operated calcium channels (SOCCs) on the plasma membrane in response to depleted internal sarco(endo)plasmic (S/ER) reticulum Ca²⁺ stores. STIM1 was initially characterized in non-excitable cells; however, evidence from both animal models and human mutations suggests a role for STIM1 in modulating Ca²⁺ homeostasis in excitable tissues as well. STIM1-dependent SOCE is particularly important in tissues undergoing sustained contraction, leading us to believe STIM1 may play a role in smooth muscle contraction. To date, the role of STIM1 in smooth muscle is unknown. In this review, we provide a brief overview of the role of STIM1-dependent SOCE in striated muscle and build off that knowledge to investigate whether STIM1 contributes to smooth muscle contractility. We conclude by discussing the translational implications of targeting STIM1 in the treatment of smooth muscle disorders.     

A potential role for differential contractility in early brain development and evolution

Differences in brain structure between species have long fascinated evolutionary biologists. Understanding how these differences arise requires knowing how they are generated in the embryo. Growing evidence in the field of evolutionary developmental biology (evo-devo) suggests that morphological differences between species result largely from changes in the spatiotemporal regulation of gene expression during development. Corresponding changes in functional cellular behaviors (morphogenetic mechanisms) are only beginning to be explored, however. Here we show that spatiotemporal patterns of tissue contractility are sufficient to explain differences in morphology of the early embryonic brain between disparate species. We found that enhancing cytoskeletal contraction in the embryonic chick brain with calyculin A alters the distribution of contractile proteins on the apical side of the neuroepithelium and changes relatively round cross-sections of the tubular brain into shapes resembling triangles, diamonds, and narrow slits. These perturbed shapes, as well as overall brain morphology, are remarkably similar to those of corresponding sections normally found in species such as zebrafish and Xenopus laevis (frog). Tissue staining revealed relatively strong concentration of F-actin at vertices of hyper-contracted cross-sections, and a finite element model shows that local contraction in these regions can convert circular sections into the observed shapes. Another model suggests that these variations in contractility depend on the initial geometry of the brain tube, as localized contraction may be needed to open the initially closed lumen in normal zebrafish and Xenopus brains, whereas this contractile machinery is not necessary in chick brains, which are already open when first created. We conclude that interspecies differences in cytoskeletal contraction may play a larger role in generating differences in morphology, and at much earlier developmental stages, in the brain than previously appreciated. This study is a step toward uncovering the underlying morphomechanical mechanisms that regulate how neural phenotypic differences arise between species.     

The role of Ca2+ in the contractility of rabbit small intestine in vitro.

This study evaluated the role of Ca2+ in spontaneous and ACh- and KCl-induced contractions in longitudinal and circular smooth muscle from rabbit small intestine in vitro. In the first experiment, the amplitude, frequency and tone of spontaneous contractions in longitudinal and circular smooth muscle of small intestine were determined and, in the second experiment, the ACh- and KCl-induced responses of longitudinal and circular smooth muscle were measured. Atropine and guanethidine reduced the amplitude and tone of contractions in longitudinal and circular muscle, but reduced the frequency of contractions in circular muscle, only. TTX attenuated the amplitude of contractions and decreased the tone of contractions in longitudinal muscle, but increased the tone in circular muscle. Ca2+-free solutions, verapamil, nifedipine and caffeine diminished the three parameters of spontaneous contractions. Thapsigargin and cyclopiazonic acid increased the amplitude and tone of contractions in ileum longitudinal muscle, only, and cyclopiazonic acid increased the amplitude of contractions in circular muscle. Ca2+-free solutions, verapamil, nifedipine, thapsigargin, cyclopiazonic acid, and caffeine diminished ACh- and KCl-induced contractions. Those results suggest that extracellular Ca2+ plays a role in spontaneous contractions, and extracellular and intracellular Ca2+ participate in the ACh- and KCl-induced contractions of rabbit small intestine.     

The determinants of contractility in the heart

The contraction-relaxation cycle of the heart represents the combined action of a variety of different components of the myocytes. For many years an ‘index’ of contractility has been sought as a means of describing and integrating the large amount of information available from the studies of heart muscle contraction. This review will undertake to show that dF/dt, recorded from the whole heart, and dT/dt, recorded in isometric studies of isolated heart muscle preparations, should not be considered as the ‘index’ of contractility. Examples will be presented in which an increasing dT/dt is paradoxically accompanied by a lower tension, while a decreasing dT/dt can occur concomitantly with an increased contractile tension. Arguments are further presented in support of the concept that Ca²⁺, in conjunction with troponin C, is the main determinant of cardiac contractility and that dT/dt reflects a dynamic equilibrium between free and troponin-bound Ca²⁺. Peak tension is thus the net result of overlapping events competing for Ca²⁺ during the latter part of contraction, that is, during Phase II of contraction as defined below. These suggestions are based upon the following considerations: (a) The Ca²⁺ pumps are active even during rest and serve to maintain low cytosolic Ca²⁺ levels, (b) As cytosolic Ca²⁺ concentration increases, Ca²⁺ pump activity also increases, (c) In addition, the Na⁺Ca²⁺ exchange is activated by elevated Ca²⁺ concentrations and serves to decrease cytosolic Ca²⁺ levels, (d) The net result is a decline in free Ca²⁺ concentration during Phase II and a reduction in the rate of cross-bridge formation until peak tension is reached. Thus, the Ca²⁺ handling elements of the myocyte serve as a finely tuned feedback device, regulating troponin C-Ca²⁺ interactions controlling the Ca²⁺ concentration of the cytosol and as a result, the actin and myosin interaction. Factors which influence the function of these elements will change the contractility of the heart.     

The role of nitric oxide in the effects of ovarian steroids on spontaneous myometrial contractility in rats

Forty ovariectomized rats were apportioned into one control and three experimental groups (n=10 each) to evaluate the role of nitric oxide in the effects of ovarian steroids on spontaneous myometrial contractility in rats. The control group (group Ov) received sesame oil once daily for 10 days, whereas rats in the experimental groups were treated with progesterone (2 mg/(rat day); group P), 17beta-estradiol (10 microg/(rat day); group E2), or progesterone and 17beta-estradiol together (group E2+P). The functionality of the arginine-nitric oxide synthase (NOS)-nitric oxide (NO) pathway in the uterine horns of sacrificed rats was evaluated in an isolated organ bath. L-Arginine, sodium nitroprusside (SNP) and 8-Br-cGMP decreased uterine contractile tension induced by electric field stimulation (EFS) in the Ov, P, and E2+P groups, but not in the E2 group. In addition, L-arginine was ineffective when applied together with a NOS inhibitor, L-nitro-N-arginine (L-NNA). The percentage of contractile inhibition was higher in the Ov and P groups compared to the E2+P group. Immunohistochemical evaluation revealed that expression of neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) in smooth muscles and nerve cells did not differ among the groups. Expression of nNOS and eNOS was strongly evident in the E2 and E2+P groups at both surface and glandular epithelium of the endometrium. iNOS expression was increased in surface epithelium of the E2 and E2+P groups. However, iNOS expression was only increased in glandular epithelial cells of the E2+P group. In conclusion, the L-arginine-NOS-NO pathway inhibits myometrial contractions via cGMP-dependent and -independent mechanisms, and while progesterone maintains the nitric oxide effects, estrogen prevents them. These results suggest that NOS does not mediate the effects of estrogen.     

The role of subcellular organelles in hormone secretion : The interactions of calcium, vitamin A, vinblastine, and cytochalasin B in PTH secretion

To determine the role of subcellular organelles in hormone secretion, we studied the interaction of low calcium concentration (low Ca), retinol (vitamin A, vit A), vinblastine (VB), and cytochalasin B (CB) in parathyroid hormone (PTH) secretion. Bovine parathyroid tissue pieces were incubated in media containing the above agents. Vit A stimulated PTH release to a mean of 170% of control. This effect of vit A was diminished when tissues were simultaneously stimulated with low Ca and, furthermore, absent when tissues were pre-incubated in low Ca.VB had no effect on low Ca-stimulated secretion, but did inhibit vit A-induced secretion in the presence of low Ca.CB stimulated PTH secretion to a mean of 150% of control during the second and third hours of incubation. CB had at least an additive effect with low Ca in stimulating PTH secretion, with a more prompt and greater response than seen in normal calcium. VB did not inhibit the acute effect of CB on secretion in normal calcium media, but did inhibit CB-induced secretion during the third hour of incubation.None of the agents stimulated the release of lysosomal cathepsin D, and vit A and CB did not stimulate the release of LDH.Our results suggest that; (1) vit A and low Ca stimulate PTH secretion through a common pathway involving the cell membrane; (2) CB stimulates PTH secretion through a separate effect on the cell membrane or submembrane microfilaments, which normally retards secretion of PTH; and (3) microtubular proteins may facilitate basal secretion of PTH, but are not involved in low Ca-stimulated secretion of PTH.     

The role of nitric oxide and l-type calcium channel blocker in the contractility of rabbit ileum in vitro

Nitric oxide (NO) and calcium channel blockers are two agents that can affect gastrointestinal motility. The goal of this work was to study the rabbit intestinal smooth muscle contraction response to (1) sodium nitroprusside (SNP), the NO donor, and its potential mechanism of action, and (2) nifedipine, the l-type Ca²⁺ channel blocker; to clarify the degree of participation by extra- and intracellular Ca²⁺ in smooth muscle contraction. We used standard isometric tension and intracellular micro-electrode recordings. To record the activity of the longitudinal smooth muscle of the ileum, segments of 1.5?cm length of the ileum were suspended vertically in organ baths of Krebs solution. The mechanical activity of the isolated ileal longitudinal muscle was recorded. Different substances were added, and the changes produced on spontaneous contraction were recorded. We found that SNP produced significant decrease, while nitric oxide synthase inhibitor produced significant increase in the amplitude of spontaneous contractions. Both apamin, the Ca²⁺-dependent K⁺ channel blocker, and methylene blue, the inhibitor of soluble guanylate cyclase, alone, partially decreased relaxation induced by SNP. Addition of both methylene blue and apamine together abolished the inhibitory effect produced by SNP on spontaneous contractions. Nifedipine produced significant decrease in the amplitude of spontaneous contractions. In conclusion, in longitudinal muscle of rabbit ileum, calcium channels blocker are potent inhibitors of spontaneous activity. However, both extracellular and intracellular Ca²⁺ participates in the spontaneous contractions. NO also has inhibitory effect on spontaneous activity, and this effect is mediated by cGMP generation system and Ca²⁺-dependent K⁺ channels.     

The action of cytochalasin A on the in vitro polymerization of brain tubulin and muscle G-actin

The presence of cytochalasin A inhibits the self-assembly of beef brain tubulin and rabbit muscle G-actin in vitro and also decreases the colchicine binding of tubulin. Prior reaction of cytochalasin A with 2-mercaptoethanol destroys its inhibitory effects. It is shown that cytochalasin A exerts its actions by reacting with sulfhydryl groups, possibly causing irreversible structural changes in the proteins. Cytochalasin B does not affect the tubulin assembly reaction.     

A spectrophotometric assay for determining the rate constants of acetylcholinesterase inhibitions.

A spectrophotometric assay procedure has been developed for determining the rate constants for the inhibition of acetylcholinesterase by carbamates and phosphates. The method permits the investigation of inhibitors over a large range in the value of the phosphorylation or carbamylation rate constants without involving the kinetic parameters of the assay substrate in the calculations.     

A novel fungal enzyme, NADPH-dependent carbonyl reductase, showing high specificity to conjugated polyketones. Purification and characterization

A novel enzyme which specifically catalyzes the reduction of conjugated polyketones was purified to homogeneity from cells of Mucor ambiguus AKU 3006. The enzyme has a strict requirement for NADPH and irreversibly reduces a number of quinones such as p-benzoquinone, alpha-naphthoquinone and acenaphthenequione. The enzyme also reduces polyketones such as isatin and ketopantoyl lactone, and their derivatives. The apparent Km values for isatin and ketopantoyl lactone are 49.9 microM and 714 microM, respectively. The reduction of ketopantoyl lactone proceeds stereospecifically to yield L-(+)-pantoyl lactone. The pro-S (A) hydrogen at C-4 of NADPH is transferred to the substrate. The enzyme is not a flavoprotein and consists of two polypeptide chains with an identical relative molecular mass of 27,500. Quercetin, dicoumarol and some SH reagents inhibit the enzyme activity. 3-Methyl-1,2-cyclopentanedione and 1,3-cyclohexanedione are uncompetitive inhibitors with Ki values of 80.9 microM and 64.5 microM, respectively, to ketopantoyl lactone.