Activation Of Smooth Muscle Contraction Research Articles

A Novel Role of Uricosuric Agent Benzbromarone in BK Channel Activation and Reduction of Airway Smooth Muscle Contraction.

The uricosuric drug benzbromarone, widely used for treatment of gout, hyperpolarizes the membrane potential of airway smooth muscle cells, but how it works remains unknown. Here we show a novel role of benzbromarone in activation of large conductance calcium-activated K+ channels. Benzbromarone results in dose-dependent activation of macroscopic big potassium (BK) currents about 1.7- to 14.5-fold with an EC50 of 111 μM and shifts the voltage-dependent channel activation to a more hyperpolarizing direction about 10 to 54 mV in whole-cell patch clamp recordings. In single-channel recordings, benzbromarone decreases single BKα channel closed dwell time and increases the channel open probability. Coexpressing β1 subunit also enhances BK activation by benzbromarone with an EC50 of 67 μM and a leftward shift of conductance-voltage (G-V) curve about 44 to 138 mV. Site-directed mutagenesis reveals that a motif of three amino acids 329RKK331 in the cytoplasmic linker between S6 and C-terminal regulator of potassium conductance (RCK) gating ring mediates the pharmacological activation of BK channels by benzbromarone. Further ex vivo assay shows that benzbromarone causes reduction of tracheal strip contraction. Taken together, our findings demonstrate that uricosuric benzbromarone activates BK channels through molecular mechanism of action involving the channel RKK motif of S6-RCK linker. Pharmacological activation of BK channel by benzbromarone causes reduction of tracheal strip contraction, holding a repurposing potential for asthma and pulmonary arterial hypertension or BK channelopathies. SIGNIFICANCE STATEMENT: We describe a novel role of uricosuric agent benzbromarone in big potassium (BK) channel activation and relaxation of airway smooth muscle contraction. In this study, we find that benzbromarone is an activator of the large-conductance Ca2+- and voltage-activated K+ channel (BK channel), which serves numerous cellular functions, including control of smooth muscle contraction. Pharmacological activation of BK channel by the FDA-approved drug benzbromarone may hold repurposing potential for treatment of asthma and pulmonary arterial hypertension or BK channelopathies.

Distinct Cellular Origins and Differentiation Process Account for Distinct Oncogenic and Clinical Behaviors of Leiomyosarcomas.

In leiomyosarcoma (LMS), a very aggressive disease, a relatively transcriptionally uniform subgroup of well-differentiated tumors has been described and is associated with poor survival. The question raised how differentiation and tumor progression, two apparently antagonist processes, coexist and allow tumor malignancy. We first identified the most transcriptionally homogeneous LMS subgroup in three independent cohorts, which we named 'hLMS'. The integration of multi-omics data and functional analysis suggests that hLMS originate from vascular smooth muscle cells and show that hLMS transcriptional program reflects both modulations of smooth muscle contraction activity controlled by MYOCD/SRF regulatory network and activation of the cell cycle activity controlled by E2F/RB1 pathway. We propose that the phenotypic plasticity of vascular smooth muscle cells coupled with MYOCD/SRF pathway amplification, essential for hLMS survival, concomitant with PTEN absence and RB1 alteration, could explain how hLMS balance this uncommon interplay between differentiation and aggressiveness.

Методы профилактики и терапии судорожного синдрома при отравлении конвульсантами холинергического ряда

Organophosphates (OPs) and carbamates are a common cause of intoxication associated with convulsive disorders. These cholinergic substances form a bond with acetylcholinesterase (AChE), thus contributing to accumulation of acetylcholine in synapses and causing typical manifestations of toxicity, including seizures. Standard antidote therapy provides sufficient symptom control, reduces seizures and decreases mortality only in case of prescription at the early stage of poisoning or preventive administration. Traditionally, atropine is used, that blocks the activity of the muscarinic cholinergic receptors in the parasympathetic nervous system and reduce the smooth muscle contraction activity, along with oximes that reactivate the reversibly inhibited AChE in the nicotinic acetylcholine receptors found in skeletal muscle. If these are not sufficient, benzodiazepines that interact with γ-aminobutyric acid receptors are used to jugulate seizures, prevent organic brain desease and post-traumatic epilepsy. There are no unified guidelines for the cases of antidotes having no effect or insufficient efficacy of antidotes. Unwanted side effects of the existing drugs and progressive decrease of efficiency within 30 min after exposure to OPs necessitate the search for new agents. Combination therapy, new dosage forms, developing original molecules or modifying the existing ones are among the developed approaches discussed in our review.

Contraction of atrial smooth muscle reduces cardiac output in perfused turtle hearts.

Unusual undulations in resting tension (tonus waves) were described in isolated atria from freshwater turtles more than a century ago. These tonus waves were soon after married with the histological demonstration of a rich layer of smooth muscle on the luminal side of the atrial wall. Research thereafter waned and the functional significance of this smooth muscle has remained obscure. Here, we provide evidence that contraction of the smooth muscle in the atria may be able to change cardiac output in turtle hearts. In in situ perfused hearts of the red-eared slider turtle (Trachemys scripta elegans), we demonstrated that activation of smooth muscle contraction with histamine (100 nmol kg-1 bolus injected into perfusate) reduced cardiac output by decreasing stroke volume (>50% decrease in both parameters). Conversely, inhibition of smooth muscle contraction with wortmannin (10 µmoll-1 perfusion) approximately doubled baseline stroke volume and cardiac output. We suggest that atrial smooth muscle provides a unique mechanism to control cardiac filling that could be involved in the regulation of stroke volume during diving.

Preparation of Pulmonary Artery Myocytes and Rings to Study Vasoactive GPCRs.

G protein-coupled receptors (GPCR) are crucial transducers of extracellular signals into changes in vascular tone. Vasoactive GPCR stimulation in the pulmonary circuit may be elicited by agonists released in acute tissue hypoxia or inflammation, as well as chronic disease. Acute responses involve activation of smooth muscle contraction or relaxation machinery causing changes in actomyosin interaction, thereby altering lumen diameter. Chronic responses may typically include activation of proliferation or fibrosis. Using pulmonary artery myocytes and pulmonary artery rings, we describe a general strategy for quantification of vasoconstrictor or vasodilator GPCR responses, and for comparison of signaling pathways in cultured cells and in contracted vessels using immunohistochemistry of contracting vessels.

RhoA and Rho-kinase inhibitors modulate cervical resistance: The possible role of RhoA/Rho-kinase signalling pathway in cervical ripening and contractility

Improper cervical function may lead premature or late-term birth. The RhoA/Rho-kinase (ROCK) signalling pathway takes part in cellular functions including smooth muscle contraction. No information is available about the cervical expression of the RhoA/ROCK system during pregnancy. Our aim was to detect the mRNA and protein expression of ROCK enzymes in rat cervices and to evaluate the effects of RhoA/ROCK inhibitors on cervical resistance.The mRNA and protein expressions of RhoA, ROCK I and II were measured in non-pregnant, pregnant and postpartum rat cervices and during parturition by Real-time qPCR and Western blot. The cervical resistance modifying effects of RhoA (simvastatin) and ROCK (fasudil, Y-27632) (10−6M) were investigated in tissue bath experiments.RhoA mRNA was increased on post-partum day 3, while the RhoA protein expression was decreased near and during parturition. ROCK I mRNA and protein expressions were fluctuating with a decrease in protein expression during parturition. ROCK II mRNA and protein expressions were sharply reduced during parturition. Simvastatin increased the cervical resistance on pregnancy days 20 and 22 while Y-27632 and fasudil reduced the resistance on pregnancy days 20.The decrease in RhoA/ROCK expression near parturition may take part in cervical ripening, especially in the final processes leading to delivery. ROCK inhibitors might be potential drug candidates to treat insufficient cervical ripening late-term pregnancies. The effect of simvastatin possibly due to its unique smooth muscle contracting activity in pregnant cervix. Compounds with simvastatin-like action might be new drug candidates for preterm cervical ripening.

Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostate.

Prostate smooth muscle contraction is critical for etiology and treatment of male lower urinary tract symptoms (LUTS) and is promoted by small monomeric GTPases (RhoA and Rac). GTPases may be activated by guanosine nucleotide exchange factors (GEFs). GEFs of the cytohesin family may indirectly activate Rac, or ADP ribosylation factor (ARF) GTPases directly. Here we investigated the expression of cytohesin family GEFs and effects of the cytohesin inhibitor Sec7 inhibitor H3 (secinH3) on smooth muscle contraction and GTPase activities in human prostate tissues. Of all four cytohesin isoforms, cytohesin-1 and -2 showed the highest expression in real-time PCR. Western blot and fluorescence staining suggested that cytohesin-2 may be the predominant isoform in prostate smooth muscle cells. Contractions induced by norepinephrine, the α1-adrenoceptor agonist phenylephrine, the thromboxane A2 analog U-46619 , and endothelin-1 and -3, as well as neurogenic contractions induced by electric field stimulation (EFS), were reduced by secinH3 (30 µM). Inhibition of EFS-induced contractions appeared to have efficacy similar to that of inhibition by the α1-adrenoceptor antagonist tamsulosin (300 nM). Combined application of secinH3 plus tamsulosin caused larger inhibition of EFS-induced contractions than tamsulosin alone. Pull-down assays demonstrated inhibition of the small monomeric GTPase ARF6 by secinH3, but no inhibition of RhoA or Rac1. In conclusion, we suggest that a cytohesin-ARF6 pathway takes part in smooth muscle contraction. This may open attractive new possibilities in medical treatment of male LUTS.

CDNA cloning and expression of Contractin A, a phospholipase A2-like protein from the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus

Venomous sea urchins contain various biologically active proteins that are toxic to predators. Contractin A is one such protein contained within the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus. This protein exhibits several biological activities, such as smooth muscle contraction and mitogenic activity. N-terminal amino acid residues of Contractin A have been determined up to 37 residues from the purified protein. In this study, we cloned cDNA for Contractin A by reverse transcription-PCR using degenerate primers designed on the basis of its N-terminal amino acid sequence. Analysis of the cDNA sequence indicated that Contractin A is composed of 166 amino acid residues including 31 residues of a putative signal sequence, and has homology to the sequence of phospholipase A2 from various organisms. In this study, recombinant Contractin A was expressed in Escherichia coli cells, and the protein was subjected to an assay to determine lipid-degrading activity using carboxyfluorescein-containing liposomes. As a result, Contractin A was found to exhibit Ca2+-dependent release of carboxyfluorescein from the liposomes, suggesting that Contractin A has phospholipase A2 activity, which may be closely associated with its biological activities.

PH-sauvagine from the skin secretion of Phyllomedusa hypochondrialis: A novel CRF-like peptide with smooth muscle contraction activity.

Amphibian skin, and particularly that of south/Central American phyllomedusine frogs, is supposed to be "a huge factory and store house of a variety of active peptides". The 40 amino acid amphibian CRF-like peptide, sauvagine, is a prototype member of a unique family of these Phyllomedusa skin peptides. In this study, we describe for the first time the structure of a mature novel peptide from the skin secretion of the South American orange-legged leaf frog, Phyllomedusa hypochondrialis, which belongs to the amphibian CRF/sauvagine family. Partial amino acid sequence from the N-terminal was obtained by automated Edman degradation with the following structure: pGlu-GPPISIDLNMELLRNMIEI-. The biosynthetic precursor of this novel sauvagine peptide, consisted of 85 amino acid residues and was deduced from cDNA library constructed from the same skin secretion. Compared with the standard sauvagine from the frog, Phyllomedusa sauvagei, this novel peptide was found to exert similar contraction effects on isolated guinea-pig colon and rat urinary bladder smooth muscle preparations.

Kinetics of Myosin Light Chain Kinase Activation of Smooth Muscle Myosin in an in Vitro Model System

During activation of smooth muscle contraction, one myosin light chain kinase (MLCK) molecule rapidly phosphorylates many smooth muscle myosin (SMM) molecules, suggesting that muscle activation rates are influenced by the kinetics of MLCK-SMM interactions. To determine the rate-limiting step underlying activation of SMM by MLCK, we measured the kinetics of calcium-calmodulin (Ca²⁺CaM)-MLCK-mediated SMM phosphorylation and the corresponding initiation of SMM-based F-actin motility in an in vitro system with SMM attached to a coverslip surface. Fitting the time course of SMM phosphorylation to a kinetic model gave an initial phosphorylation rate, kp(o), of ~1.17 heads s⁻¹ MLCK⁻¹. Also, we measured the dwell time of single streptavidin-coated quantum dot-labeled MLCK molecules interacting with surface-attached SMM and phosphorylated SMM using total internal reflection fluorescence microscopy. From these data, the dissociation rate constant from phosphorylated SMM was 0.80 s⁻¹, which was similar to the kp(o) mentioned above and with rates measured in solution. This dissociation rate was essentially independent of the phosphorylation state of SMM. From calculations using our measured dissociation rates and Kd values, and estimates of SMM and MLCK concentrations in muscle, we predict that the dissociation of MLCK from phosphorylated SMM is rate-limiting and that the rate of the phosphorylation step is faster than this dissociation rate. Also, association with SMM (11-46 s⁻¹) would be much faster than with pSMM (<0.1-0.2 s⁻¹). This suggests that the probability of MLCK interacting with unphosphorylated versus phosphorylated SMM is 55-460 times greater. This would avoid sequestering MLCK to unproductive interactions with previously phosphorylated SMM, potentially leading to faster rates of phosphorylation in muscle.

MLCK Induces Actin Filament Bundling and Moves on Bundled Actin and Stress Fibers of Smooth Muscle Cells

We are interested in the mechanism of phosphorylation of smooth muscle myosin (SMM) by the myosin light chain kinase -calmodulin-Ca2+ complex (MLCK-CaM-Ca2+). This reaction is required for activation of SMM catalytic activity and smooth muscle contraction. We have been studying the interactions between MLCK, actin, SMM and smooth muscle stress fibers at a single molecule level using total internal reflectance fluorescence microscopy (TIRF). We observed that MLCK induces actin filament bundling. The number and length of the bundles depends upon the MLCK concentration and incubation time. MLCK also induced actin-tropomyosin complex (actin-Tm) bundling, but with longer bundle length. The N-terminal 1-75 peptide of MLCK (GST-N1-75 MLCK), which has three DFRxxL actin binding motifs does not cause actin or actin-Tm bundling, suggesting that other actin binding domains on MLCK are required for bundling. These findings may suggest that MLCK plays a role in stabilizing stress fibers in smooth muscle cells.With quantum dots labeled MLCK (QD-MLCK), we have observed that MLCK not only causes actin bundling, but moves along the actin bundles and actin-Tm bundles. In a more physiological system, skinned human airway smooth muscle cells, direct observations of single QD-MLCK molecules show clearly that MLCK co-localizes with and can move along the actin- and myosin-containing stress fibers, at high ionic strength, or at physiological ionic strength with CaM-Ca2+ and ATP. GST-N1-75 MLCK also moves along smooth muscle cell stress fibers. The diffusion coefficient, calculated from mean-squared-displacement (MSD) data from MLCK-QDs' trajectories, indicates that the mechanism by which one MLCK phosphorylates multiple SMMs may involve MLCK movement along thin and/or thick filaments on a time scale measured in seconds.

MLCK moves on Actin Filament and Stress Fiber of Smooth Muscle Cells

We are interested in the mechanism of phosphorylation of smooth muscle myosin (SMM) by the myosin light chain kinase -calmodulin-Ca2+ complex (MLCK-CaM-Ca2+). This reaction is required for activation of SMM catalytic activity and smooth muscle contraction. In previous studies we characterized tightly-bound SMM-MLCK-CaM complexes in an in vitro model system and demonstrated that SMM-MLCK-CaM complexes co-purified from smooth muscle were functional, i.e. MLCK was able to phosphorylate SMM and the phosphorylated SMM resulted in actin filament motility in an in vitro assay.

Effects of Resveratrol, a Grape Polyphenol, on Uterine Contraction and Ca2+Mobilization in Ratsin Vivoandin Vitro

Dysmenorrhea is directly related to elevate prostaglandin F (PGF)(₂α) levels. In Western medicine, this condition is treated using nonsteroidal antiinflammatory drugs. Because nonsteroidal antiinflammatory drugs produce many side effects, Chinese medicinal therapy is considered as a feasible alternative for treating dysmenorrhea. Many special physiological components used in Chinese medicine, such as resveratrol, have been isolated and identified. Resveratrol has many physiological functions, such as antioxidation and anticarcinogenic effects. However, the relationship between uterine smooth muscle contraction and resveratrol remains unknown. Here, we studied the in vitro and in vivo effects of resveratrol on uterine smooth muscle contraction. The uterus was separated from a female Sprague Dawley rat, and uterine smooth muscle contraction activity was measured and recorded. The results demonstrated that 1) resveratrol treatment inhibited PGF(₂α)-, oxytocin-, acetylcholine-, and carbachol-induced uterine contractions in rats; 2) resveratrol inhibited uterine contractions stimulated by the Ca²(+) channel activator (Bay K 8644) and depolarization in response to high K(+) (KCl); 3) resveratrol inhibited PGF(₂α)-induced increases in the [Ca²(+)]i in human uterine smooth muscle cells; 4) resveratrol could mimic Ca²(+) channel blockers to block Ca²(+) influx through voltage-operated Ca²(+) channels in the plasma membrane; and 5) resveratrol inhibited PGF(₂α)-induced uterine contractions in rats in vivo. Resveratrol inhibited uterine contractions induced by PGF(₂α) and high K(+) in a concentration-dependent manner in vitro; furthermore, it inhibited Ca²(+)-dependent uterine contractions. Thus, resveratrol consistently suppressed the increases in intracellular Ca²(+) concentrations ([Ca²(+)]i) induced by PGF(₂α) and high K(+) concentrations. It can be assumed that resveratrol probably inhibited uterine contraction by blocking external Ca²(+) influx, leading to decreased [Ca²(+)]i. Therefore, resveratrol can be considered as a feasible alternative therapeutic agent for dysmenorrhea.

The In Vitro Study of Resveratrol on Uterine Contraction and Ca2+ Mobilization in the Rat.

Dysmenorrhea is directly related to elevated PGF2alpha levels. It is treated with NSAIDs (nonsteroid antiinflammatory drugs) in Western medicine. Since NSAIDs produce many side effects, Chinese medicinal therapy is considered as a feasible alternative medicine. Many special physiological components (ex: Resveratrol) in Chinese medicine have been isolated and identified. Resveratrol have a lot of physiological functions like anti-oxidation and anti-cancer effects. However, the relationship between uterine smooth muscle contraction and resveratrol remains veiled. We studied the in vitro effects of resveratrol on uterine smooth muscle contraction. The uterus was separated from female SD rat and uterine smooth muscle contraction activity was measured and recorded. Resveratrol inhibited uterine contractions induced by PGF2alpha, Ca2+ channel activator Bay K 8644, and high K+ in a concentration-dependent manner in vitro; furthermore, resveratrol inhibited the Ca2+-dependent uterine contractions. Thus, resveratrol consistently suppressed the increases in intracellular Ca2+ concentrations ([Ca2+]i) induced by PGF2alpha and high K+. Thus, resveratrol probably inhibited uterine contraction by blocking external Ca2+ influx, leading to a decrease in [Ca2+]i. Thus, resveratrol may be considered as a feasible alternative therapeutic agent for dysmenorrhea. (poster)

Rho-associated kinase plays a role in rabbit urethral smooth muscle contraction, but not via enhanced myosin light chain phosphorylation

The involvement of Rho-associated kinase (ROK) in activation of rabbit urethral smooth muscle contraction was investigated by examining the effects of two structurally distinct inhibitors of ROK, Y27632 and H1152, on the contractile response to electric field stimulation, membrane depolarization with KCl, and α1-adrenoceptor stimulation with phenylephrine. Both compounds inhibited contractions elicited by all three stimuli. The protein kinase C inhibitor GF109203X, on the other hand, had no effect. Urethral smooth muscle strips were analyzed for phosphorylation of three potential direct or indirect substrates of ROK: 1) myosin regulatory light chains (LC20) at S19, 2) the myosin-targeting subunit of myosin light chain phosphatase (MYPT1) at T697 and T855, and 3) cofilin at S3. The following results were obtained: 1) under resting tension, LC20 was phosphorylated to 0.65±0.02 mol Pi/mol LC20 (n=21) at S19; 2) LC20 phosphorylation did not change in response to KCl or phenylephrine; 3) ROK inhibition had no effect on LC20 phosphorylation in the absence or presence of contractile stimuli; 4) under resting conditions, MYPT1 was partially phosphorylated at T697 and T855 and cofilin at S3; 5) phosphorylation of MYPT1 and cofilin was unaffected by KCl or phenylephrine; and 6) KCl- and phenylephrine-induced contraction-relaxation cycles did not correlate with actin polymerization-depolymerization. We conclude that ROK plays an important role in urethral smooth muscle contraction, but not via inhibition of MLCP or polymerization of actin.

Renal calcium‐sensing receptors influence renal hemodynamics via nitric oxide‐mediated vasodilation

Calcium (Ca) is a cofactor in both activation of nitric oxide synthase (NOS) and vascular smooth muscle contraction. Both endothelial cells and vascular smooth muscle express Ca‐sensing receptors (CaSR), which increase intracellular Ca in response to increased extracellular Ca. Ca mimetics enhance this signaling. We hypothesized that the Ca mimetic Cinacalcet (Amgen) would cause NO‐mediated renal vasodilation. The effect of Cinacalcet on blood pressure (BP) and renal blood flow (RBF) without and with NOS inhibition by L‐NAME (10 mg/kg bw) were measured in Inaction‐anesthetized male Sprague‐Dawley rats. Without L‐NAME, an i.v. bolus of 30 mg/kg bw Cinacalcet increased BP from 102 ±5 to 112 ±4 mmHg (p&lt;0.001) within 3 min and RBF increased 15% from 6.41 ±0.4 to 7.38 ± 0.4 ml/min/gkw (p&lt;0.001). L‐NAME increased BP by 29 ±4 mmHg and reduced RBF by 45% (p&lt;0.001). Then, Cinacalcet administration produced a similar pressor response as before (11 ±2 mmHg, p&lt;0.01), but now RBF remained unchanged. Thus, we observed the systemic response to activating CaSR was pressor and NO‐independent, but the renal vasodilation was NO‐mediated. We conclude that activating the CaSR does cause NO‐mediated renal vasodilation. This dissociation of systemic and renal responses suggests that in the renal vasculature, CaSR‐activated NOS activity may be a particularly important factor in maintaining renal perfusion.

Integrin-linked Kinase Regulates N-WASp-mediated Actin Polymerization and Tension Development in Tracheal Smooth Muscle

The contractile stimulation of smooth muscle tissues stimulates the recruitment of proteins to membrane adhesion complexes and the initiation of actin polymerization. We hypothesized that integrin-linked kinase (ILK), a beta-integrin-binding scaffolding protein and serine/threonine kinase, and its binding proteins, PINCH, and alpha-parvin may be recruited to membrane adhesion sites during contractile stimulation of tracheal smooth muscle to mediate cytoskeletal processes required for tension development. Immunoprecipitation analysis indicted that ILK, PINCH, and alpha-parvin form a stable cytosolic complex and that the ILK.PINCH.alpha-parvin complex is recruited to integrin adhesion complexes in response to acetylcholine (ACh) stimulation where it associates with paxillin and vinculin. Green fluorescent protein (GFP)-ILK and GFP-PINCH were expressed in tracheal muscle tissues and both endogenous and recombinant ILK and PINCH were recruited to the membrane in response to ACh stimulation. The N-terminal LIM1 domain of PINCH binds to ILK and is required for the targeting of the ILK-PINCH complex to focal adhesion sites in fibroblasts during cell adhesion. We expressed the GFP-PINCH LIM1-2 fragment, consisting only of LIM1-2 domains, in tracheal smooth muscle tissues to competitively inhibit the interaction of ILK with PINCH. The PINCH LIM1-2 fragment inhibited the recruitment of endogenous ILK and PINCH to integrin adhesion sites and prevented their association of ILK with beta-integrins, paxillin, and vinculin. The PINCH LIM1-2 fragment also inhibited tension development, actin polymerization, and activation of the actin nucleation initiator, N-WASp. We conclude that the recruitment of the ILK.PINCH.alpha-parvin complex to membrane adhesion complexes is required to initiate cytoskeletal processes required for tension development in smooth muscle.

Leukotrienes as mediators of skin inflammation.

Leukotrienes (LTs) derived from the 5-lipoxygenase pathways of arachidonic acid metabolism, are a new group of biologically active mediators. LTC4, LTD4 and LTE4 are considered collectively to account for the activity of slow reacting substance of anaphylaxis (SRS-A). They have potent smooth muscle contracting activity (Drazen et al., 1980; Holme et al., 1980) and cause vascular permeability changes in guinea-pig skin (Peck, Piper & Williams, 1981). LTB4 has been shown to be a potent chemotactic and chemokinetic agent for polymorphonuclear leucocytes (PMNs) (Ford-Hutchinson et al., 1980; Smith, Ford-Hutchinson & Bray, 1980) and to cause vascular permeability changes in rabbit, rat and guinea-pig skin (Bray et al., 1981a). In human skin LTB4 has been shown to be a chemotactic agent for neutrophils (Bray, Ford-Hutchinson & Smith, 1981b; Camp et al., 1982). LTC4 and LTD4 have pronounced inflammatory actions in human skin causing weal and flare responses and increases in blood flow at low concentrations (Camp et al., 1982; Bisgard, Kristensen & Sondergaard, 1982). The present studies were designed to investigate a variety of leukotrienes on permeability responses in rabbit and guinea-pig skin.

The functional effects of a c‐kit tyrosine inhibitor on guinea‐pig and human detrusor

To describe the effect of a specific c-kit receptor inhibitor (imatinib mesylate) on human detrusor strips in vitro and guinea-pig cystometry in vivo, and to show histological data suggesting differences in the distribution of interstitial cells of Cajal (ICC)-like cells in 'normal' and overactive human detrusor, as these cells have been identified as possible mediators of spontaneous activity and excitability in bladder smooth muscle. Specimens of human detrusor were stained immunohistochemically with a c-kit antibody. Human detrusor strips were mounted in a superfused organ-bath apparatus, and smooth muscle contraction was evoked with carbachol and electrical field stimulation in the presence and absence of imatinib mesylate. Also, guinea-pig urodynamic studies were conducted before and after i.v. administration of imatinib mesylate, and changes in bladder variables and spontaneous activity were recorded. Imatinib mesylate (10(-6)M) inhibited evoked smooth muscle contraction and spontaneous activity in overactive human detrusor, with less effect on normal human tissue. Imatinib mesylate (10(-5)M) improved bladder capacity, compliance, voided volumes, urinary frequency, and reduced contraction thresholds and spontaneous activity during guinea-pig cystometry. c-kit labelling showed significantly more ICC-like cells in overactive human detrusor than in normal specimens. c-kit receptor blockers have inhibitory effects on guinea-pig and overactive human detrusor, possibly via c-kit receptors on bladder ICC-like cells. This and the possibility that there are more ICC-like cells in overactive bladder suggest that the c-kit receptor may provide a novel target for treating detrusor overactivity.

Regulation of smooth muscle contraction by calcium, monomeric GTPases of the Rho subfamily and their effector kinases.

A key event in the activation of smooth muscle contraction ist the phosphorylation of the regulatory light chains of myosin (r-MLC) at Ser19 which is predominantly catalyzed by the Ca2+ and calmodulin dependent myosin light chain kinase, MLCK (Gallager et al., 1997). Recently, it has been suggested that, in addition, r-MLC phosphorylation and contraction may be induced in a Ca2+-independent manner by several protein kinases, such as Rho associated kinase, ZIP kinase, and integrin linked kinase, ILK (reviewed in Ganitkevich et al, 2002). These kinases may, hence, be involved in Ca2+-independent contractions (Kureishi et al., 1999) leading to an increased Ca2+-sensitivity of r-MLC phosphorylation and contraction. Although the physiological role of these kinases is far from clear, they may be of importance during the maintained phase of a contraction when intracellular [Ca2+] has returned to near resting values (Himpens and Somlyo, 1988; Lucius et al., 1998). One goal of this study, therefore, was to test whether Ca2+ is required for tension maintenance of an agonist induced contraction using the membrane permeant form of the caged Ca-chelator, diazo2.