Bitter Taste Receptors as Regulators of Abdominal Muscles Contraction.

Bitter taste receptors (TAS2R) are expressed in many non-sensor tissues including skeletal muscles but their function remains unexplored. The aim of this study is to investigate the role of TAS2R in rat abdominal skeletal muscles contractions using denatonium, a TAS2R agonist. Low concentration of denatonium (0.01 mmol/l) caused a significant decrease of amplitudes of the electrical field stimulation (EFS)-induced contractions in abdominal skeletal muscles preparations in vitro. This inhibitory effect was significantly reduced when the preparations were pre-incubated with gentamicin (0.02 mmol/l) used as a non-specific inhibitor of IP3 formation or with BaCl(2) (0.03 mmol/l) applied to block the inward-rectifier potassium current. All experiments were performed in the presence of pipecuronium in order to block the nerve stimulation of the contractions. The data obtained suggest that denatonium decreases the force of rat abdominal muscles contractions mainly via activation of TAS2R, phosphatidylinositol 4,5-biphosphate and its downstream signal metabolites.

Ghrelin signaling in human mesenteric arteries.

The hypothesis is that the ghrelin signal pathway consists of new participants including a local second mediator in human mesenteric arteries. The contractile force of isometric artery preparations was measured using a wire-myograph. Whole-cell patch clamp experiments were performed on freshly isolated single smooth muscle cells from the same tissue. After the addition of ghrelin (100 nmol) the outward potassium currents conducted through iberiotoxin-sensitive calcium-activated potassium channels with a large conductance were almost entirely abolished. The effect of ghrelin on potassium currents was insensitive to selective inhibitors of cAMP-dependent protein kinase and soluble guanylate cyclase, but was eliminated in the presence of des-octanoyl ghrelin and O-(octahydro-4,7-methano-1H-inden-5-yl) carbonopotassium dithioate (D-609). Ghrelin dose-dependently increased the force of contraction of native, endothelium-denuded and mostly of endothelium-denuded and treated with tetrodotoxin human mesenteric arteries preconstricted with 1 nmol endothelin-1. This effect of ghrelin was blocked when the bath solution contained 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), 4-amino-5-(4-methylphenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2), D-609, 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203x), pertussis toxin, 2-aminoethyl diphenylborinate (2-APB), indomethacin, (5Z,13E)-(9S,11S,15R)-9,15,Dihydroxy-11-fluoro-15-(2-indanyl)-16,17,18,19,20,pentanor-5,13-prostadienoic acid (AL-8810) - a non-selective prostanoid receptor antagonist, 5-(4-Chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethyl pyrazolo (SC-560) - a selective cyclooxygenase 1 inhibitor, ozagrel - a selective thromboxane A(2) synthase inhibitor or T prostanoid receptor antagonist GR32191B. It is concluded that ghrelin increases the force of contraction of human mesenteric arteries by a novel mechanism that involves Src kinase, mitogen-activated protein kinase kinase (MEK), cyclooxygenase 1 and T prostanoid receptor agonist, most probably thromboxane A(2).

Ghrelin signalling in guinea-pig femoral artery smooth muscle cells.

AIM: Our aim was to study the new signalling pathway of ghrelin in the guinea-pig femoral artery using the outward I(K) as a sensor. METHODS: Whole-cell patch-clamp experiments were performed on single smooth muscle cells, freshly isolated from the guinea-pig femoral artery. The contractile force of isometric preparations of the same artery was measured using a wire-myograph. RESULTS: In a Ca2+- and nicardipine-containing external solution, 1 mmol L(-1) tetraethylammonium reduced the net I(K) by 49 +/- 7%. This effect was similar and not additive to the effect of the specific BK(Ca) channel inhibitor iberiotoxin. Ghrelin (10(-7) mol L(-1)) quickly and significantly reduced the amplitudes of tetraethylammonium- and iberiotoxin-sensitive currents through BK(Ca) channels. The application of 5 x 10(-6) mol L(-1) desacyl ghrelin did not affect the amplitude of the control I(K) but it successfully prevented the ghrelin-induced I(K) decrease. The effect of ghrelin on I(K) was insensitive to selective inhibitors of cAMP-dependent protein kinase, soluble guanylyl cyclase, cGMP-dependent protein kinase or a calmodulin antagonist, but was effectively antagonized by blockers of BK(Ca) channels, phosphatidylinositol-phospholipase C, phosphatidylcholine-phospholipase C, protein kinase C, SERCA, IP(3)-induced Ca2+ release and by pertussis toxin. The ghrelin-induced increase in the force of contractions was blocked when iberiotoxin (10(-7) mol L(-1)) was present in the bath solution. CONCLUSIONS: Ghrelin reduces I(K(Ca)) in femoral artery myocytes by a mechanism that requires activation of Galpha(i/o)-proteins, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C, protein kinase C and IP(3)-induced Ca2+ release.

[Urocortin decreases phosphorylation of MYPT1 and increases the myosin phosphatase activity via elevation of the intracellular level of cAMP].

Urocortin, a peptide hormone related to the corticotropin releasing factor, is suggested to be involved in blood pressure regulation by dilating the peripheral blood vessels. In rat tail arteries, urocortin-induced vasodilation is due to a decrease in myofilament Ca2+ sensitivity the mechanism of which is still unclear. In this study, the hypothesis was tested that the decrease in Ca2+ sensitivity in mouse tail arteries results from the activation of myosin light chain phosphatase. The relaxation of KCl-precontracted (42 mM) intact mouse tail arteries by urocortin (1 nM and 10 nM) was significantly inhibited by 1 microM antisauvagine30, a CRF-2 receptor antagonist (p < 0.05, n = 3). The addition of 1 microM KT 5720, an inhibitor of PKA, to intact rat tail arteries did not affect the KCl-induced force but significantly attenuated the urocortin-induced relaxation (n = 5). In alpha-toxin permeabilized mouse tail arteries, urocortin relaxed submaximally activated preparations at constant pCa 6.1 by 37.6 +/- 8.2% (n = 5) as compared to control vessels (n = 5, p < 0.001). The relaxation in permeabilized vessels was inhibited by pre-treatment with 30 microM Rp-8-CPT-cAMPS, an inactive analogue of cAMP. In permeabilized mouse tail arteries, treatment with 100 nM urocortin was associated with dephosphorylation of MLC20(Ser19) and MYPT1(Thr696/Thr850). The effect of urocortin on MYPTI dephosphorylation was completely abolished by 30 M Rp-8-CPT-cAMPS and mimicked by the cAMP analogue Sp-5,6-DCI-cBiMPS. Based on these findings, we propose that the urocortin-induced relaxation is due to a decrease in calcium sensitivity mediated by a cAMP-dependent increase in the activity of MLCP.

HO-1 induction in the guinea-pig stomach: protection of smooth muscle functional performance during cobalt-induced oxidative stress.

Distribution and enzymatic activity of haem oxygenase (HO) was investigated in the stomach of healthy guinea pigs or animals subjected to in vivo cobalt-induced oxidative stress. The physiological role of HO-1 and HO-2 isoenzymes in fundic and antral area of the stomach was assessed by studying the action of HO substrate--hemin--on ionic currents of single smooth muscle cells. The data obtained suggest that HO-1 induction might serve in the guinea-pig stomach as genetically determined defense mechanism aimed to combat toxic stress-related pathology in order to preserve the functional performance of the organ.

Characterization of voltage-gated calcium currents in freshly isolated smooth muscle cells from rat tail main artery.

The aim of the present study was to characterize voltage-gated Ca2+ currents in smooth muscle cells freshly isolated from rat tail main artery in the presence of 5 mmol L(-1) external Ca2+. Calcium currents were identified on the basis of their voltage dependencies and sensitivity to nifedipine, Ni2+ and cinnarizine. In the majority of the cells studied, T- and L-type currents were observed, while the remaining cells showed predominantly L-type currents. In the latter group of cells, holding potential change from -50 to either -70 or -90 mV increased the corresponding inward current amplitude while its voltage activation threshold remained unchanged. The steady state inactivation of L-type Ca2+ channels showed half-maximal inactivation at -38 mV. A Ca2+-dependent inactivation was also evident. Nifedipine (3 micromol L(-1)) blocked L-type but not T-type Ca2+ currents. Ni2+ (50 micromol L(-1)) as well as cinnarizine (1 micromol L(-1)) suppressed the nifedipine-resistant, T-type component of the currents. At higher concentrations, both Ni2+ (0.3-1 mmol L(-1)) and cinnarizine (10 micromol L(-1)) blocked the net inward current. Replacement of Ca2+ with 10 mmol L(-)1 Ba2+ significantly increased the amplitude of L-type Ca2+ currents. These results demonstrate that smooth muscle cells freshly isolated from rat tail main artery may be divided into two populations, one expressing both L- and T-type and the other only L-type Ca2+ channels. Furthermore, this report shows that in arterial smooth muscle cells cinnarizine potently inhibited T-type currents at low concentrations (1 micromol L(-1)) but also blocked L-type Ca2+ currents at higher concentrations (10 micromol L(-1)).

Thyrotropin-releasing hormone activates KCa channels in gastric smooth muscle cells via intracellular Ca2+ release.

Thyrotropin-releasing hormone (TRH) is released in high concentrations into gastric juice, but its direct effect on gastric smooth muscles has not been studied yet. We undertook studies on TRH effect on gastric smooth muscle using contraction and patch clamp methods. TRH was found to inhibit both acetylcholine- and BaCl2-induced contractions of gastric strips. TRH, applied to single cells, inhibited the voltage-dependent Ca2+ currents and activated the whole-cell K+ currents. The TRH-induced changes in K+ currents and membrane potential were effectively abolished by inhibitors of either intracellular Ca2+ release channels or phospholipase C. Neither activators, nor blockers of protein kinase C could affect the action of TRH on K+ currents. In conclusion, TRH activates K+ channels via inositol-1,4,5-trisphosphate-induced release of Ca2+ in the direction to the plasma membrane, which in turn leads to stimulation of the Ca2+-sensitive K+ conductance, membrane hyperpolarization and relaxation. The data imply that TRH may act physiologically as a local modulator of gastric smooth muscle tone.

Urocortin hyperpolarizes stomach smooth muscle via activation of Ca2+-sensitive K+ currents.

The effect of urocortin (Uro), a recently discovered neuropeptide with selectivity towards corticotropin-releasing hormone type 2 receptor, was tested on whole cell currents expressed by guinea-pig gastric antrum smooth muscle cells. Uro (1 pmol/l-1 nmol/l) caused a concentration-dependent increase of Ca2+-sensitive K currents (I(K)) up to 500% as compared to control currents and did not affect the kinetics and voltage-dependence of inward Ca2+ currents. The I(K)-increasing effect of Uro was fully antagonized by preliminary emptying of intracellular Ca2+ stores with ryanodine and cyclopiazonic acid, as well as by bath application of selective blockers of adenylyl cyclase and cAMP-dependent protein kinase (PKA), but not by inhibitors of guanylyl cyclase, cGMP-dependent protein kinase, and protein kinase C. Comparable I(K) increase was obtained by forskolin (activator of adenylyl cyclase), Sp-cAMPS (activator of PKA), or by intracellular application of the catalytic subunit of PKA. It was concluded that Uro binds to a selective receptor in antral smooth muscle cells where it stimulates I(K) via PKA-dependent increase of Ca2+ concentration near the plasma membrane due to enhanced release from intracellular calcium stores.

Two populations of smooth muscle cells in the guinea-pig gastric antrum.

K+ outward currents (I[K]) expressed by guinea-pig antral smooth muscle cells were studied using the whole-cell voltage-clamp technique. In about 88% of cells depolarization steps applied from Vh = -70 mV activated a fast transient component (I[K(to)]) with voltage-dependent characteristics, and a noninactivating component with slow activation kinetics (I[K(sl)]). Both components were carried by K+ ions. Apamin (10 nM to 1 microM) selectively depressed I(K[to]) in a concentration-dependent manner. I(K(sl)) was blocked by 1 mM tetraethylammonium or 0.1 microM charybdotoxin. 10 mM tetraethylammonium abolished both components of I(K). Nicardipine (1 microM) did not affect the voltage- and time-dependent characteristics of the net I(K), but reduced the current density of I(K[sl]) from 22.36+/-1.38 microA/cm2 to 13.06+/-0.92 microA/cm2 at +40 mV. In about 12% of the cells depolarization-evoked I(K) could be separated as two pharmacologically distinguishable components: a glipizide-sensitive current (forming about 70% of the net I[K]) and a charybdotoxin-sensitive current (30% of the net I[K]). Nicardipine (1 microM) affected neither the amplitude nor the time-course of I(K) of this cell population. The depletion of intracellular Ca2+ stores by thapsigargin (1 microM) or ryanodine (1 microM) led to a 50-200% increase of I(K[sl]) in the majority of cells and to an about 30% increase of the net I(K) in 12% of cells. The data obtained suggest the existence of at least two populations of cells in guinea-pig antral smooth muscle. Twelve percent of cells seem to be responsible for the generation of slow wave potentials, while 88% of cells most probably respond passively to the electrotonically spread depolarization.

Oxytocin-induced changes in single cell K+ currents and smooth muscle contraction of guinea-pig gastric antrum.

To study the effects of oxytocin on both spontaneous phasic contractions and K+ outward currents (IK) of the so-called 'non-target' smooth muscle cells, physiological concentrations of oxytocin ranging between 10(-12) mol/l and 10(-8) mol/l were applied to smooth muscle preparations and single voltage-clamped cells isolated from the circular layer of the guinea-pig gastric antrum. Oxytocin (10(-12) mol/l to 10(-8) mol/l) suppressed, in a dose-dependent manner, the tetrodotoxin- and atropine-resistant spontaneous phasic contractions and shifted rightward the dose-response curves of 10(-7) mol/l charybdotoxin and 10(-3) mol/l BaCl2. In cells with preloaded intracellular Ca2+ stores, oxytocin (10(-12) mol/l to 10(-9) mol/l) caused a dose-dependent activation of the charybdotoxin-blockable non-inactivating component of IK (IK(sl)) of single voltage-clamped cells, which was accompanied by hyperpolarization of the cell membranes. 8Lys-vasopressin and 8arg-vasopressin failed to mimic the effects of oxytocin on both contraction and K+ currents. Further, the oxytocin-induced activation of IK(sl) was effectively antagonized by 5 x 10(-8) mol/l U-73122 or 5 x 10(-6) mol/l 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (inhibitors of the cell membrane phospholipase C), as well as by intracellularly applied heparin (selective inhibitor of inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release channels). In cells incubated in the absence of Ca2+ entry throughout the study, oxytocin (10(-9) mol/l) caused a slight and transient increase of IK(sl) amplitudes. Neither ryanodine (10(-6) mol/l) nor cyclopiazonic acid (10(-6) mol/l) were able to restore the IK-activating effect of oxytocin in these cells. The data obtained suggest (i) that selective oxytocin receptors are present on the membranes of guinea-pig antral smooth muscle cells, (ii) that the oxytocin-related relaxation may result from the activation of Ca(2+)-sensitive K+ conductivity via activation of IP3-induced release of Ca2+ from the submembrane located cisternae of the sarcoplasmic reticulum Ca2+ stores and (iii) in turn, this evokes a non-inactivating component of IK, hyperpolarizing the cell membrane.

Two components of potassium outward current in smooth muscle cells from the circular layer of human jejunum.

Two components of the outward K+ currents (Ik) of cells isolated from the circular layer of human jejunum were investigated using the conventional whole-cell voltage clamp method. A fast transient Ik component could only be elicited by depolarization in cells dialysed with pipette solution of pCa < 7.4. This Ik component was strongly voltage dependent, and could be selectively abolished by 30 mumol/l quinidine. Its amplitudes decreased in the absence of Ca entry, the decrease depending on the duration of cell exposure to media containing calcium-blockers, and disappeared after depletion of intracellular Ca2+ stores. The steady-state component of Ik was sensitive to tetraethylammonium. This component had comparable amplitudes at pCa = 8.4 or pCa = 7.4 of the pipette solution, and was present during a long-lasting exposure of cells to solutions containing Ca(2+)-blocking drugs.

Main components of voltage-sensitive K+ currents of the human colonic smooth muscle cells.

The aim of the present study is to characterize voltage-sensitive macroscopic K+ current (IK) components in freshly isolated human colonic smooth muscle cells. IK components were studied by the conventional whole-cell voltage clamp method. We found two main components of IK. A transient IK component with fast kinetics, IK(fi), activated upon a holding potential of Vh = -80 mV and inactivated completely following a 4-second-long prepulse to 0 mV. IK(fi) was abolished by Vh = -50 mV, as well as by a pipette solution containing 11 mM EGTA. The second, non-inactivating IK component, IK(ni), had comparable amplitudes at Vh = -80 mV and Vh = -50 mV and could not be inactivated completely, even by positive conditioning stimuli. The amplitudes of IK(ni) depended strongly on the Ca2+ entry, while the amplitudes and the time course of IK(fi) were modulated mainly by the intracellular Ca2+ concentration. About 80% of IK(ni) was selectively inhibited by 0.5 microM apamin. IK(fi) was insensitive to apamin and was totally abolished by 20 mM tetraethyl ammonium extracellularly. According to their voltage dependencies, inactivation properties and pharmacological sensitivity to various K+ channels antagonists, these IK components differ from those described in colonic smooth muscle cells of laboratory animals.

Ca(2+)-induced Ca2+ release activates K+ currents by a cyclic GMP-dependent mechanism in single gastric smooth muscle cells.

The participation of sarcoplasmic reticulum Ca2+ release channels in the activation of Ca(2+)-sensitive K+ currents (IK(Ca)) by cyclic dibutyryl GMP was investigated in smooth muscle cells from the circular layer of guinea-pig gastric fundus. All experiments were performed in the presence of 3 microM nicardipine into the bath and low Ca2+ buffering capacity of the pipette-filling solution (pCa 7.4). Ruthenium red (10 microM) as well as its combination with 10 microM heparin abolished the cyclic GMP-induced activation of IK(Ca), while 10 microM heparin remained ineffective. Ryanodine (10 microM) and the subsequently added 1 microM thapsigargin induced a relatively small increase in IK(Ca) amplitudes. The addition of 10 microM ryanodine to 1 microM thapsigargin-containing bath solution caused a vast increase in IK(Ca). It is hypothesyzed that protein kinase G-induced vectorial Ca2+ flux from the cell bulk and sarcoplasmic reticulum Ca2+ stores toward the plasma membrane is realized by a spontaneous Ca(2+)-induced Ca2+ release from a superficially situated Ca2+ store.

Cyclic GMP-induced activation of potassium currents by sarcoplasmic reticulum Ca2+ pump-dependent mechanism.

In voltage-clamped single smooth muscle cells from the circular layer of the guinea-pig gastric fundus NO-liberating substance or an analogue of cyclic 3, 5'-guanosine monophosphate (cGMP) increased or decreased the outward K+ current amplitudes depending on the Ca2+ buffering capacity of the intracellular medium. In a high EGTA-containing pipette solution dibutyryl-cGMP or sodium nitroprusside (SNP) attenuated both the fast and the late K+ current components. In pipette solution with lower Ca2+ -buffering capacity these drugs caused a sustained increase of K+ current amplitudes, which was effectively antagonized by thapsigargin, an inhibitor of Ca2+ -ATPase in the sarcoplasmic reticulum (SR). Our data suggest that, in gastric fundus smooth muscles, NO-liberating substances and cGMP analogues contribute to the activation of a Ca2+ -release mechanism from the cell bulk, i.e. the myoplasm surrounding the contractile filaments, towards the plasma membrane, crossing the SR Ca2+ -stores. Thus, a decreased intracellular free calcium concentration ([Ca2+]) is coupled with an elevation of subplasmalemmal calcium, which in turn causes cell membrane hyperpolarization. The latter is a consequence of the opening of tetraethylammonium-sensitive Ca2+ -activated K+ channels and leads to sustained smooth muscle relaxation, most characteristic for gastric fundus preparations.

L-type calcium channels may fill directly the IP3-sensitive calcium store.

The relationship between inositol-1,4,5-trisphosphate-sensitive Ca2+ stores and Ca2+ entry through potential-dependent L-type Ca2+ channels was examined using whole-cell voltage-clamp technique in cells of longitudinal muscle layer of guinea-pig ileum. It was found that heparin (10(-10) mol/l) in the pipette rapidly inhibited the current through L-type Ca2+ channels. Neither an inhibitor of the sarcoplasmic reticulum Ca2+ pump (cyclopiazonic acid) nor blockers of Ca(2+)-induced Ca2+ release (ryanodine or ruthenium red) affected the Ca2+ current. The failure of heparin to affect Ca(2+)-currents through L-type Ca2+ channels in cells from circular muscle of the same organ suggested that heparin had no direct effect on L-type Ca2+ channels. Thus the inhibition of the latter in heparin-loaded cells from the longitudinal layer is supposed to be Ca(2+)-dependent due to the overfilling of the inositol-1,4,5-trisphosphate-sensitive Ca2+ store.

Inhibition of Ca2+ current in ileal cells by cyclopiazonic acid and ryanodine.

The effects of cyclopiazonic acid and its combination with ryanodine on the inward Ca2+ current (ICa) were investigated in smooth muscle cells isolated from the circular layer of guinea-pig ileum. The ICa of these cells exhibited two components: a low-threshold, nicardipine (5 microM)-resistant, fast-inactivating component and a high-threshold, nicardipine-blockable and slowly inactivating component. Neither cyclopiazonic acid (up to 10 microM) nor ryanodine (10 microM) was able to affect both these components of ICa, when applied separately. Cyclopiazonic acid and ryanodine combination led to total abolishment of the high-threshold component, leaving the low-threshold component unaffected. The data presented suggest a process of Ca(2+)-dependent inactivation of the high-threshold component, elicited by an increase in the subplasmalemmal Ca2+ concentration due to Ca2+ released from the sarcoplasmic reticulum. It is considered that the combination of cyclopiazonic acid and ryanodine can be used as a valuable method to study the calcium sensitivity of both components of the ICa.

Two types of Ca(2+)-channels in cells from the circular layer of guinea-pig ileum.

In this paper data are presented concerning some morphometrical characteristics and passive electrical properties of cell membranes from the circular layer of guinea-pig ileum (CC). The main purpose of the study has been to analyze the inward calcium current in these cells. Using the whole-cell voltage-clamp method the presence of T-like type of calcium channels (conducting low-threshold, fast inactivated, strictly potential-dependent and dihydropyridine-resistant calcium inward current--If) could be found in membranes of the cells investigated. The participation of If in the total inward current of CC was deduced from the presence of a low-threshold "hump" in the current-voltage relation curve (I/V-curve) between -80 and -50 mV, and also from a negative shift of this curve at holding potential Vh = -90 mV, compared to the one obtained at Vh = -50 mV. Besides, in the presence of a dihydropyridine Ca2+ antagonist, a fast inactivating component of the inward current could be recorded, which was effectively blocked by 0.3 mmol/l Ni2+ from outside. According to Tsien's criteria (1983) our data suggest the coexistence of T- and L-types calcium channels in CC.

Participation of calcium, released from the IP3-sensitive Ca-store in activation of Ca-dependent potassium conductance of ileal smooth muscle cells.

The apamin-, charybdotoxin- (CTX) and glibenclamide- (GLB) sensitive components, which form the active part of the net potassium outward current (IK) in single smooth muscle cells from the longitudinal layer of guinea-pig ileum (LC), were investigated for their sensitivity to calcium. The experiments were carried out by the whole-cell voltage-clamp method. A successful block of all Ca-sources (with heparin and nifedipine; heparin and cyclopiazonic acid while the intracellular Ca-concentration--[Ca2+]i--was kept at 3 x 10(-8) mol/l by 11 mmol/l EGTA into the pipette solution) led to the complete inhibition of IK. The deeply located Ca-sensitive Ca-pool was effectively isolated by the high concentration of the chelator, which was proved by the fact that ruthenium red and ryanodine failed to affect IK. The GLB-sensitive component of IK demonstrated Ca-gated properties, while both the other components were activated most probably by calcium, released form the IP3-sensitive Ca-pool. It was concluded that the IP3-induced Ca-release mechanism plays an important role in the regulation of K(+)-conductivity in LC.