Guinea-pig Taenia Coli Research Articles

Contractile action of Mn2+ via Ca2+ channels activated by bay K 8644 in guinea-pig taenia coli.

Mn2+ has been shown to inhibit K+-induced contraction of smooth-muscle, to induce contraction of smooth-muscle in Ca2+-free, K+ medium and to activate the contractile proteins of skinned fibres of smooth muscle cells. Further work has suggested that Mn2+ penetrates the cytoplasm through voltage-dependent Ca2+ channels when the cell membranes of smooth muscles are depolarized with K+. We have investigated whether in Ca2+-free medium, Mn2+ enters the cytoplasm through Ca2+ channels and induces contraction of guinea-pig taenia coli in the presence of Bay K 8644, a dihydropyridine Ca2+-channel agonist which prolongs the open state of the voltage-dependent Ca2+ channels in smooth-muscle cells. In Ca2+-free medium the application of 5 mM Mn2+ in the presence of Bay K 8644 caused contraction of and concomitant increase in Mn2+ uptake in guinea-pig taenia coli smooth muscle. In the presence of Bay K 8644 nifedipine, a dihydropyridine Ca2+ channel antagonist, dose-dependently inhibited both manganese uptake and the contraction induced by Mn2+. These results suggest that Mn2+ enters the cytoplasm through dihydropyridine-sensitive, voltage-dependent Ca2+ channels activated by Bay K 8644 and then induces contraction in taenia coli.

Effect of Mn2+ on the development of tension induced in guinea-pig taenia coli by Bay K 8644.

We have studied the effects of Mn2+ on the contractile response induced by Bay K 8644, a dihydropyridine Ca2+ agonist, on guinea-pig taenia coli. Mn2+ (5 mM) completely inhibited Bay K 8644 (10(-6) M)-induced rhythmic contraction and contracture to baseline values in normal Ca2+ medium; thereafter, the contraction progressively increased to about 90% of the K+ (60 mM)-induced tonic response. In Ca2+-free medium in the presence of Bay K 8644 Mn2+ also evoked contraction and a concomitant increase in Mn2+ influx into the cytoplasm. These results suggest that during the opening of voltage-dependent Ca2+ channels activated by Bay K 8644, Mn2+ can enter cytoplasm through the channels and induce contraction in taenia coli.

Role of cAMP, cGMP, and protein kinase C in regulation of calcium current through the L-type calcium channels in the electroexcitable membrane of smooth muscle cells

Regulation of calcium current through L-type calcium channels (I Ca,L) of the guinea pigtaenia coli smooth muscle cell (SMC) membrane by cyclic nucleotides and protein kinase C (PKC) was studied using a voltage-clamp technique with intracellular dialysis or membrane patch perforation with amphotericin B. Non-selective blockers of serine/threonine kinase, staurosporine and H-7 reduced theI Ca,L amplitude in a dose-dependent manner. Dose-dependent suppression ofI Ca,L was also produced by a selective PKC blocker, chelerythrine, and a cAMP-and cGMP-dependent protein kinase (PKA, PKG), blocker H-8. Forskolin, which increases the intracellular level of cAMP, as well as membrane-permeant cAMP analogs, dibutyryl-cAMP (db-cAMP) and 8-bromo-cAMP, exerted complex effects onI Ca,L. The latter increased at their concentrations below 10 μM and decreased at their higher concentrations. 8-Bromo-cGMP reducedI Ca,L in all cases. Addition of 50 μM GTPγS to the micropipette solution caused a marked and slowly developing increase inI Ca,L. 8-Bromo-cAMP (1 μM) increasedI Ca,L by 30%, both in the control and during the action of GTPγS. The blockade of PKC by 10 μM chelerythrine removed the effect of GTPγS onI Ca,L. The results suggest that basal activity of L-type calcium channels in SMC of the guinea pigtaenia coli depends on PKC- and PKA-dependent phosphorylation. PKC can increase theI Ca,L amplitude provided G proteins are activated. cAMP at low concentrations likewise increasesI Ca,L (probably through activation of PKA). PKG apparently mediatesI Ca,L drops evoked by cAMP at high concentrations and by cGMP.

P2-receptor antagonists: IV. Blockade of P2-receptor subtypes and ecto-nucleotidases by compounds related to reactive blue 2.

Effects of reactive blue 2 and twelve structurally related compounds were studied on contractions of the rat vas deferens elicited by alpha,beta-methylene ATP (alpha,beta-MeATP; mediated by P2X-receptors), relaxations of the carbachol-precontracted guinea-pig taenia coli elicited by adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS; mediated by P2Y-receptors), and the degradation of ATP by rat vas deferens tissue. All compounds, except acid blue 41 and acid blue 129 (at up to 100 microM), shifted the concentration-response curve of alpha,beta-MeATP in the rat vas deferens to the right. Most increased, but uniblue A greatly decreased, the maximum of the curve. In the case of cibacron blue 3GA and reactive blue 19, of which three concentrations were tested, the Arunlakshana-Schild regression was linear, and the slope did not differ from unity. The apparent Kd values of the effective substances ranged between 0.7 and 111 microM. Most compounds increased the contraction of the rat vas deferens elicited by high K+. In the guinea-pig taenia coli, all compounds, except uniblue A and reactive blue 19 (at up to 100 microM), shifted the concentration-response curve of ADPbetaS to the right in a parallel manner. In the case of acid blue 129 and acid blue 80, of which three concentrations were tested, the slope of the Arunlakshana-Schild regression did not differ from unity. The apparent Kd values of the effective substances were between 0.7 and 69 microM. Most compounds also reduced the relaxation of the guinea-pig taenia coli elicited by noradrenaline. The removal of ATP from the medium by vas deferens tissue was decreased only by reactive blue 2, cibacron blue 3GA, uniblue A and reactive blue 19, with IC25% values between 17 and 62 microM. The structure-activity relationships for P2X- and P2Y-receptor blockade in this series are strikingly dissimilar. In reactive blue 2 and its isomers, for example, both the 1-amino-anthraquinone-2-sulphonate core and the 'side-chain' of the molecule are involved in P2X-receptor binding; P2Y-receptor affinity, in contrast, resides largely or totally in the anthraquinone core. The most promising antagonists are uniblue A which is P2X- versus P2Y-selective and acid blue 129 which is P2Y- versus P2X-selective, both with few, if any, non-P2-receptor effects at concentrations blocking the respective P2-subtype.

P-675 - The inhibitory effects of amlexanox on carbachol-induced contractions in colored rabbit ciliary smooth muscle and guinea pig taenia coli

P-675 - The inhibitory effects of amlexanox on carbachol-induced contractions in colored rabbit ciliary smooth muscle and guinea pig taenia coli

O-38 - Contractile action by Mn2+ through Ca2 + channel activated by Bay K 8644 in guinea-pig taenia coli

O-38 - Contractile action by Mn2+ through Ca2 + channel activated by Bay K 8644 in guinea-pig taenia coli

P-679 - Effects of sodium nitroprusside on muscle tension, intracellular Ca2+ level, myosin phosphorylation and ionic current in guinea-pig taenia coli

P-679 - Effects of sodium nitroprusside on muscle tension, intracellular Ca2+ level, myosin phosphorylation and ionic current in guinea-pig taenia coli

P-677 - Effects of W-7, trifluoperazine and cyclopiazonic acid on phasic contraction induced by high K+ in guinea-pig taenia coli

P-677 - Effects of W-7, trifluoperazine and cyclopiazonic acid on phasic contraction induced by high K+ in guinea-pig taenia coli

Temperature sensitivity of force and shortening velocity in maximally activated skinned smooth muscle.

We have studied the temperature dependence of isometric force, rate of force development and maximal shortening velocity (Vmax) in skinned guinea-pig taenia coli smooth muscle. To eliminate the influence of temperature on activation mechanisms, maximally thiophosphorylated preparations were used. Isometric force in the range 2-35 degrees C was maximal at 22 degrees C with a decrease of 25% at 2 degrees C and 10% at 35 degrees C. Rate of tension development from rigor after photolytic release of ATP increased four-fold between 5 degrees C and 30 degrees C. Vmax increased with a Q10 of about 2 (1.6, range 5-15 degrees C, and 2.2, range 22-30 degrees C). The temperature dependence of the rate of tension development indicates rate-limitation by transitions into force-generating states or by the hydrolysis reaction. The temperature dependence of Vmax reflects effects of temperature on reactions (e.g. the ADP-release) associated with cross-bridge detachment. The small temperature dependence of steady-state force in smooth compared with skeletal muscle suggests differences in the cross-bridge reactions controlling the number of attached force-generating states in the two muscle types.

Exogenous caldesmon promotes relaxation of guinea-pig skinned taenia coli smooth muscles: inhibition of cooperative reattachment of latch bridges?

In smooth muscle, the state of prolonged contraction (latch state) is associated with very slow energy turnover and cycling of crossbridges that are dephosphorylated. A similar state may be reproduced in skinned fibres when the calcium-induced contraction is terminated by calcium removal with ethylenebis(oxonitrilo)tetraacetate (EGTA) and, during the slow relaxation that follows, force is maintained by dephosphorylated crossbridges that cycle slowly or not at all and may cooperatively reattach after detachment (Khromov et al. 1995, Biophys J 69:2611-2622). In guinea-pig skinned taenia coli that has been pretreated by prolonged incubation with caldesmon (5 microM), the rate of relaxation is approximately 1.6 times greater than in untreated controls, with half-times of relaxation being 1.3 and 2.1 min, respectively. In contrast, preloading the fibres with calponin does not accelerate relaxation. Preloading the fibres with caldesmon also accelerates the relaxation of skinned fibres from the state of rigor contraction when the latter is terminated by immersion into an ATP-containing relaxing solution or, in the presence of inorganic phosphate (Pi), also by flash-photolytic release of ATP from caged-ATP. Even in the latter case, relaxation is comparatively slow, possibly because of cooperative reattachment of dephosphorylated crossbridges which delays net crossbridge detachment and hence relaxation. We propose that by inhibition of cooperative reattachment caldesmon accelerates relaxation, even in the presence of Pi, and that the latch-like state of skinned fibres is supported by dephosphorylated cooperatively attaching crossbridges and may be regulated by the activity of caldesmon in the smooth muscle cell.

Antagonism of P2X receptors in guinea-pig vas deferens by diinosine pentaphosphate

Diinosine pentaphosphate (Ip 5I) antagonized contractions, mediated via P2X receptors, evoked by diadenosine pentaphosphate (Ap 5A) and ATP in the guinea-pig isolated vas deferens with pA 2 values of 6.4±0.17 (10 d.f.) and 6.5±0.10 (10 d.f.), respectively. Ip 5I (30 μM) did not affect contractile responses evoked by noradrenaline. Ip 5I (up to 100 μM) did not antagonize P2Y receptors in the guinea-pig taenia coli, nor P1 or P2 receptors in the guinea-pig left atrium.

Components of depolarization-induced transmembrane ion current in isolated smooth muscle cells of the guinea pigtaenia coli

Transmembrane ion currents in isolated single smooth muscle cells (SMC) from the guinea pigtaenia coli were investigated using a whole-cell mode of the patch-clamp technique. Currents induced by depolarizing shifts in the membrane potential from its holding level of −60 mV contained an initial inward phase (Ca2+ current), which in 30–40 msec was followed by an outward phase. It was shown that outward current was carried by K ions and consisted at least of three components: one Ca2+-independent K+ current of delayed rectifier (KV) and two Ca2+-dependent K+ currents. The latter can be further divided into the apamin-sensitive (SK) and charybdotoxin-sensitive (BK) currents. It was found that relative contributions of these three components in total outward current at 0 mV were 35–45%, 5–15%, and 45–55% for KV, SK, and BK currents, respectively. A potential-dependent current carried by Ci ions was also found. This Cl− current had inward direction within the range of potentials below the chloride equilibrium potential (E Cl) and outward direction above theE Cl. The magnitude of Cl− current was significantly lower than the magnitude of total K+ current.

Stellettamide-A, a novel inhibitor of calmodulin, isolated from a marine sponge.

1. Stellettamide A (ST-A), a novel marine toxin isolated from a marine sponge, inhibited high K+(72.7 mM)-induced contraction in the smooth muscle of guinea-pig taenia coli with an IC50 of 88 microM. 2. In the taenia permeabilized with Triton X-100, ST-A inhibited Ca2+ (3 and 10 microM)-induced contractions with an IC50 of 46 microM for 3 microM Ca2+ and 105 microM for 10 microM Ca2+. In the permeabilized taenia, calyculin-A (300 nM), a potent inhibitor of type-1 and type-2A phosphatases, induced sustained contraction in the absence of Ca2+. ST-A had no effect on this contraction. 3. ST-A inhibited Mg2+-ATPase activity in native actomyosin prepared from chicken gizzard with an IC50 of 25 microM. 4. In a reconstituted smooth muscle contractile system containing calmodulin, myosin light chain (MLC) and MLC kinase, ST-A inhibited MLC phosphorylation with an IC50 of 152 microM. The inhibitory effect of ST-A was antagonized by increasing the concentration of calmodulin. 5. ST-A inhibited calmodulin activity, assessed by Ca2+/calmodulin-dependent enzymes, (Ca2+-Mg2+)-ATPase of erythrocyte membrane, with an IC50 of 100 microM and phosphodiesterase prepared from bovine cardiac muscle with an IC50 of 52 microM. The inhibitory effect on phosphodiesterase activity was antagonized by increasing the calmodulin concentration. 6. Interaction between ST-A and calmodulin was demonstrated by instantaneous quenching of the intrinsic tyrosine fluorescence of calmodulin by ST-A (3-300 microM). Similar results were obtained in the presence or absence of Ca2+ suggesting that ST-A binds to calmodulin and that Ca2+ is not essential for the binding of ST-A to calmodulin. 7. These results suggest that ST-A, isolated from marine metabolites, is a novel inhibitor of calmodulin.

Receptor-mediated diacylglycerol kinase translocation dependent on both transient increase in the intracellular calcium concentration and modification by protein kinase C

Diacylglycerol kinase (DG kinase) is activated by various stimuli in many types of cells. We reported earlier that carbachol (CCh) induced DG kinase translocation from the cytosolic fraction to the membrane fraction in guinea pig taenia coli ( Biochem. Pharmacol., 50: 591–599, 1995). In this study, the regulation mechanisms of DG kinase translocation are reported, based on the following findings: 1) CCh sustained an increase in DG kinase in the membrane fraction and a decrease in the cytosolic fraction; 2) blocking calcium influx by removing extracellular calcium did not affect the CCh-induced sustained DG kinase translocation; 3) exposing purified protein kinase C (PKC) to DG kinase increased DG kinase affinity to octylglycoside micelles only with the enzyme extracted from the cytosolic fraction; and 4) CCh-induced DG kinase translocation was reversed by removing CCh, and the serine/threonine phosphatase inhibitor, okadaic acid, blocked the reversal of the translocation. These results suggest that CCh-induced DG kinase translocation is promoted by both a transient increase in intracellular calcium, which may be released from the intracellular store, and by DG kinase phosphorylation by PKC.

Endothelin-activated calcium signaling in enteric glia derived from neonatal guinea pig.

The ability of guinea pig enteric glia to respond to endothelins was examined using fura 2-based digital microscopy in glial cells derived from guinea pig taenia coli. Each isoform of endothelin (ET-1, ET-2, ET-3) evoked dose-dependent and equipotent increases in intracellular Ca2+ concentration ([Ca2+]i) and in percentage of cells responding, 4alaEt-1, an ETB receptor agonist, elicited similar [Ca2+]i increments. BQ-788, an ETB antagonist, inhibited [Ca2+]i responses to endothelin. Preincubation of glia with U-73122 a phospholipase C inhibitor, abolished the [Ca2+]i response to ET-3 exposure. Thapsigargin also eliminated ET-3-evoked Ca2+ signaling. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, introduced into glial cells by radio frequency electroporation, blocked [Ca2+]i responses to ET-3 (100 nM) in 63% of glia. Sustained elevation in [Ca2+]i was abolished by removal of Ca2+ from the buffer and inhibited 85. -3% by Ni2+ (1 mM). Preincubation of glia with 100 nM phorbol 12-myristate 13-acetate (24 h) also inhibited sustained increments in [Ca2+]i by 87%. The presence of IP3 receptors in enteric glia was confirmed by immunofluorescent confocal microscopy.

Evidence that NO acts as a redundant NANC inhibitory neurotransmitter in the guinea-pig isolated taenia coli.

1. The relative contribution of the putative transmitters, nitric oxide (NO) and an apamin-sensitive factor, possibly ATP, to inhibitory responses evoked by electrical field stimulation (EFS; 0.2-5 Hz, 0.2 ms duration, supra-maximal voltage for 10 s) of non-adrenergic, non-cholinergic (NANC) nerves was investigated in the guinea-pig isolated taenia coli contracted with histamine (1 microM). 2. Peak relaxations to EFS (0.2-5 Hz) were tetrodotoxin (1 microM)-sensitive, maximal at 0.2 Hz and completely resistant to the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG; 100 microM) in either the presence or absence of atropine (1 microM). Furthermore, the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ; 10 microM), the cytochrome P450 inhibitor and free radical generator, 7-ethoxyresorufin (7-ER; 10 microM) and the NO scavenger, oxyhaemoglobin (HbO; 30 microM) had no effect on EFS-induced relaxations alone and in combination with L-NOARG (100 microM). 3. Maximum relaxation to the NO donor, sodium nitroprusside (SNP; 1 microM) was significantly reduced by HbO (30 microM), abolished by 7-ER (10 microM) and ODQ (10 microM) but was unaffected by apamin (0.1 microM), an inhibitor of small conductance Ca(2+)-activated K+ channels. 4. The relaxation to EFS at 0.2 Hz was resistant to apamin but those to 0.5 and 5 Hz were significantly reduced. EFS (0.2-5 Hz)-evoked relaxations that persisted in the presence of apamin were further significantly inhibited by L-NOARG (100 microM) or ODQ (10 microM), but not by HbO (30 microM) or 7-ER (10 microM). 5. ATP (1-30 microM) produced concentration-dependent relaxations that were abolished by apamin (0.1 microM), unaffected by ODQ (10 microM) but only significantly reduced by L-NOARG (100 microM) at the lowest concentration of ATP (1 microM) used. 6. Nifedipine (0.3 microM), abolished contractions to 67 mM KCl, histamine (10 microM), endothelin-1 (0.03 microM), 5-hydroxytryptamine (5-HT; 10 microM) and the thromboxane-mimetic, 9-11-dideoxy-9 alpha, 11 alpha-methano-epoxy-prostaglandin F2 alpha (U46619; 0.1 microM). 7. The findings of the present study suggest that NO is released during NANC nerve stimulation, but plays no role in NANC relaxations in the guinea-pig taenia coli unless the effects of another apamin-sensitive, nerve-derived hyperpolarizing factor (NDHF) are blocked. Thus, we propose that in this tissue, NO acts as a 'backup' or redundant NANC nerve inhibitory transmitter and like NDHF mediates relaxation via hyperpolarization.

Polymerization of myosin on activation of rat anococcygeus smooth muscle.

The in vivo state of assembly of myosin in vertebrate smooth muscle is controversial. In vitro studies on purified smooth muscle myosin show that it is monomeric (10S) under relaxing conditions and filamentous under contraction conditions. Electron microscopic and antibody labelling studies of intact smooth muscles, on the other hand, suggest that myosin is filamentous in the relaxed as well as the contracting state and that 10S myosin occurs only in trace amounts. However, birefringence, conventional EM and X-ray diffraction evidence suggests that in certain smooth muscles in vivo (e.g. rat anococcygeus), while myosin filaments exist in the relaxed state, their number increases on contraction. Here, we have used low temperature electron microscopic techniques (rapid freezing followed by freeze-substitution), which preserve labile components in close to their in vivo state, to detect any change in filament number on contraction. The results from rat anococcygeus have been compared with those from guinea pig taenia coli, in which other techniques have revealed no change in filament number. In the anococcygeus, we find evidence for a 23% increase in filament density in transverse sections of contracting muscle compared with relaxed muscle. In the taenia coli we find no change. These results are in qualitative agreement with earlier findings. They provide evidence for polymerization of myosin in contracting rat anococcygeus, and suggest that this process is subtle and occurs only in some smooth muscles.

Action of FMRFamide-like Peptides on Porcine Gastrointestinal Motility In Vitro

Decker, B., B. Vadokas, U. Kutschenreuter, K. Golenhofen, K. Voigt, G. P. Mcgregor and K. Mandrek. Action of FMRFamide-like peptides on porcine gastrointestinal motility in vitro. Peptides 18(10) 1531–1537, 1997.—Mechanical activity was recorded in circular and longitudinal smooth muscle preparations isolated from extensive regions of the porcine gastrointestinal tract in response to the FMRFamide-like neuropeptides F8Famide and A18Famide. In all preparations, the peptides were about equipotent in producing phasic contractions or enhancing spontaneous activity. The most prominent responses were observed in jejunal longitudinal strips which were on the average 91% (±4% SEM, n = 15; 10 −6 M) of the histamine (10 −5 M) responses. The peptide-induced phasic activity was completely abolished by nifedipine but was unaffected by tetrodotoxin, atropine, phentolamine, yohimbine, phenoxybenzamine, propranolol, methysergide, cimetidine, indomethacin, levallorphane or naloxone. Both peptides enhanced acetylcholine-induced contractions. However, bovine ileum and guinea-pig taenia coli was not affected by these peptides. The results indicate that F8F- and A18F-amide contract porcine gastrointestinal smooth muscle by acting directly via non-opioid receptors on L-type calcium channels. In addition an increase of the sensitivity to cholinergic stimulation occurs.

P-658 - ATP release evoked by sitmuration of peptides-receptors froe taenia coli and vas deferens of guinea pig

P-658 - ATP release evoked by sitmuration of peptides-receptors froe taenia coli and vas deferens of guinea pig

Vanadate oxidation activates contraction in skinned smooth muscle without myosin light chain phosphorylation.

Phosphorylation of the myosin regulatory light chain (LC20-P1) is the major route of smooth muscle activation. However, after prior exposure to vanadate, permeabilized guinea pig taenia coli smooth muscle contracts in the absence of LC20-P1. We characterized the vanadate-induced contraction and investigated the mechanism of this novel activation pathway. Addition of vanadate to a control contracture (6.6 microM Ca2+) inhibits force (effective dose for 50% response was approximately 100 microM). In contrast, preincubation with high concentrations of vanadate (threshold at 1-2 mM) elicited a contraction on subsequent transfer of the fiber to a vanadate-free, Ca(2+)-free solution. Maximum isometric force of approximately 60% of control was obtained in fibers preincubated in 4 mM vanadate for 10 min. Addition of Ca2+ to a vanadate-induced contracture increased force, but the total force never exceeded the initial control. After maximal thiophosphorylation of LC20 with adenosine 5'-O-(3-thiotriphosphate), treatment with vanadate did not increase force. Unloaded shortening velocity (Vmax) was similar in Ca2+ and vanadate contractures and was additive. After thiophosphorylation, preincubation in vanadate had no effect on Vmax, suggesting that vanadate affected the number of activated bridges and not cycle rate. Vanadate mechanisms likely involve oxidation, since preincubation with 4 mM vanadate and 25 mM dithiothreitol (DTT) did not produce force. DTT could reverse a vanadate-induced contracture in 30-60 min. Subsequently, fibers demonstrated control contraction/relaxation cycles. Thus vanadate treatment did not cause irreversible damage, such as the extraction of proteins. Potential oxidation sites are proteins at 17 kDa and between 30 and 40 kDa, which were not alkylated by N-ethylmaleimide if they were treated in the presence of vanadate or in the rigor state. Vanadate-induced contractures are likely mediated by a reversible oxidation that activates cross bridges similarly to that of LC20-Pi and may play an important role in oxidant injury.