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.

Beta1-subunit of the Ca2+-activated K+ channel regulates contractile activity of mouse urinary bladder smooth muscle.

1. The large-conductance calcium-activated potassium (BK) channel plays an important role in controlling membrane potential and contractility of urinary bladder smooth muscle (UBSM). These channels are composed of a pore-forming alpha-subunit and an accessory, smooth muscle-specific, beta1-subunit. 2. Our aim was to determine the functional role of the beta1-subunit of the BK channel in controlling the contractions of UBSM by using BK channel beta1-subunit 'knock-out' (KO) mice. 3. The beta-galactosidase reporter (lacZ gene) was targeted to the beta1 locus, which provided the opportunity to examine the expression of the beta1-subunit in UBSM. Based on this approach, the beta1-subunit is highly expressed in UBSM. 4. BK channels lacking beta1-subunits have reduced activity, consistent with a shift in BK channel voltage/Ca2+ sensitivity. 5. Iberiotoxin, an inhibitor of BK channels, increased the amplitude and decreased the frequency of phasic contractions of UBSM strips from control mice. 6. The effects of the beta1-subunit deletion on contractions were similar to the effect of iberiotoxin on control mice. The UBSM strips from beta1-subunit KO mice had elevated phasic contraction amplitude and decreased frequency when compared to control UBSM strips. 7. Iberiotoxin increased the amplitude and frequency of phasic contractions, and UBSM tone of UBSM strips from beta1-subunit KO mice, suggesting that BK channels still regulate contractions in the absence of the beta1-subunit. 8. The results indicate that the beta1-subunit, by modulating BK channel activity, plays a significant role in the regulation of phasic contractions of the urinary bladder.

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)).

Low levels of K(ATP) channel activation decrease excitability and contractility of urinary bladder.

Activation of ATP-sensitive potassium (K(ATP)) channels can regulate smooth muscle function through membrane potential hyperpolarization. A critical issue in understanding the role of K(ATP) channels is the relationship between channel activation and the effect on tissue function. Here, we explored this relationship in urinary bladder smooth muscle (UBSM) from the detrusor by activating K(ATP) channels with the synthetic compounds N-(4-benzoylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropionamide (ZD-6169) and levcromakalim. The effects of ZD-6169 and levcromakalim on K(ATP) channel currents in isolated UBSM cells, on action potentials, and on related phasic contractions of isolated UBSM strips were examined. ZD-6169 and levcromakalim at 1.02 and 2.63 microM, respectively, caused half-maximal activation (K1/2) of K(ATP) currents in single UBSM cells (see Heppner TJ, Bonev A, Li JH, Kau ST, and Nelson MT. Pharmacology 53: 170-179, 1996). In contrast, much lower concentrations (K(1/2) = 47 nM for ZD-6169 and K1/2 = 38 nM for levcromakalim) caused inhibition of action potentials and phasic contractions of UBSM. The results suggest that activation of <1% of K(ATP) channels is sufficient to inhibit significantly action potentials and the related phasic contractions.

Control of the phasic and tonic contractions of guinea pig stomach by a ryanodine-sensitive Ca2+ store.

In some smooth muscle cells, the rise in intracellular Ca2+ as a result of a Ca2+ influx via plasma membrane Ca2+ channels can activate a further increase in intracellular Ca2+ as a result of Ca2+ release from intracellular stores. This study examined the role of the Ca2+-induced Ca2+ release from the ryanodine-sensitive intracellular Ca2+ stores in shaping the smooth muscle contractions of guinea pig stomach. The contractile activity of isolated muscle strips of the fundus, corpus and antrum region of the stomach was recorded under isometric conditions. Ryanodine, an activator of Ca2+-induced Ca2+ release, concentration dependently (10(-7)-3x10(-5) M) increased the tone of fundus and corpus strips. Ryanodine had a dual action on the phasic contractions of the antrum and corpus: increase by the low concentrations (up to 10(-6) M) and inhibition by the high concentrations (10(-6)-3x10(-5) M). Nifedipine (10(-5) M) completely inhibited the ryanodine (10(-6) M)-induced phasic contractions and only partly the ryanodine (3x10(-5) M)-induced tonic contractions. In the presence of 10(-5) M cyclopiazonic acid, a specific inhibitor of sarcoplasmic reticulum Ca2+-ATPase, ryanodine (3x10(-5) M) further increased the tone of the corpus and fundus strips. Ryanodine (3x10(-5) M) induced tonic contractions in the fundus and corpus precontracted by acetylcholine (10(-5) M), and inhibited the acetylcholine (10(-6) M)-induced phasic contractions in the antrum and corpus. Ruthenium red, an inhibitor of Ca2+-induced Ca2+ release, concentration dependently (10(-6)-10(-4) M) decreased the tone and amplitude of the phasic contractions. The data obtained provide evidence for the participation of a sarcoplasmic reticulum Ca2+-induced Ca2+ release mechanism in shaping the tonic and phasic contractions of guinea pig stomach, and highlight important tissue differences.

The role of a ryanodine-sensitive Ca(2+)-store in the regulation of smooth muscle tone of the cat gastric fundus.

The smooth muscle of the gastric fundus maintains spontaneous tone, but the mechanism underlying this activity is not fully understood. The aim of the present study was to examine whether Ca2+ release from the sarcoplasmic reticulum (SR) could play a role in the maintenance of the spontaneous smooth muscle tone of the cat gastric fundus. The effects on the contractile activity of SR Ca2+ release activators ryanodine and caffeine and of the inhibitor ruthenium red were studied. The contractile activity of isolated muscle strips was recorded under isometric conditions using organ baths. Ryanodine concentration-dependently (10(-7)-3.10(-5) mol/l) increased the tone of the fundus strips. In the presence of nifedipine (10(-5) mol/l), ryanodine induced a nifedipine-resistant tonic contraction. The tonic contraction induced after inhibition of the SR Ca(2+)-pump by cyclopiazonic acid was potentiated by ryanodine (10(-5) mol/l). In strips precontracted with supramaximal concentration of acetylcholine (10(-5) mol/l), ryanodine (3.10(-5) mol/l) further potentiated the tone. Caffeine (10(-4)-10(-2) mol/l) decreased and even completely inhibited the tone, suggesting some other effects of caffeine. Ruthenium red concentration-dependently (10(-6)-10(-4) mol/l) decreased the tone. The present data provide evidence for the role of Ca2+ release from a SR ryanodine-sensitive Ca2+ store in the maintenance of the muscle tone of the cat gastric fundus.

Myorelaxant activity of 2-t-butyl-4-methoxyphenol (BHA) in guinea pig gastric fundus.

This study investigates the mechanism whereby the antioxidant 2-t-butyl-4-methoxyphenol (BHA) relaxes guinea pig gastric fundus smooth muscle. In circular smooth muscle strips, 10 microM cyclopiazonic acid, a specific inhibitor of sarcoplasmic reticulum Ca2+-ATPase, induced a prolonged rise in tension which depended on the presence of extracellular Ca2+. BHA (pIC50 = 5.83), sodium nitroprusside (6.85), isoproterenol (7.69) and nifedipine (8.02), but not 2,6-di-t-butyl-4-methoxyphenol (DTBHA) (up to 30 microM), relaxed muscle strips contracted with cyclopiazonic acid. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyri dine-5-carboxylate (Bay K 8644) (1 microM) antagonised the nifedipine- but not the BHA-induced relaxation. Nifedipine and isoproterenol (10 microM) caused a decrease in spontaneous tone, but did not counteract the subsequent rise in tension elicited by 10 microM cyclopiazonic acid. Conversely, 100 microM BHA and 100 microM sodium nitroprusside not only significantly reduced spontaneous tone but also markedly impaired the response of the muscles to cyclopiazonic acid. DTBHA failed to show either effect. When added to preparations completely relaxed by 100 microM BHA, 10 mM tetraethylammonium still elicited nifedipine-sensitive tonic and phasic contractions in the presence or absence of 10 microM cyclopiazonic acid. BHA and DTBHA inhibited, in a concentration-dependent manner, the Ca2+-promoted contraction of strips depolarised by 10 mM tetraethylammonium. The BHA antagonism showed a non-competitive profile while that of DTBHA was competitive. In muscle strips at rest, 10 microM BHA caused a significant increase in tissue cAMP concentration, leaving cGMP unmodified. To conclude, the myorelaxant action of BHA on gastric fundus smooth muscle appears to be mediated partly by an increase in cAMP levels and partly by inhibition of Ca2+ influx from the extracellular space.

Role of sarcoplasmic reticulum in the myorelaxant activity of nitric oxide donors in guinea pig gastric fundus.

The relaxant effect of two nitric oxide (NO) donors: sodium nitroprusside and 3-morpholino-sydnonimine (SIN-1) on circular smooth muscle strips isolated from guinea pig gastric fundus was studied with the view to elucidating the mechanism, which underlies the NO-induced relaxation of this tissue. Both sodium nitroprusside (10(-9)-10(-5) M) and SIN-1 (10(-9)-10(-4) M) suppressed the spontaneous fundus tone and hyperpolarized the muscle cells by about 5 mV. They antagonized the acetylcholine (10(-6) M)-induced tone and exerted their relaxant effects even when Ca2+ influx into the cells was triggered through the Na+/Ca2+ exchanger. Sodium nitroprusside and SIN-1 antagonized the contraction induced by cyclopiazonic acid (10(-5) M), a specific inhibitor of the sarcoplasmic reticulum Ca2+-ATPase. In the presence of high concentrations of sodium nitroprusside or SIN-1, cyclopiazonic acid (10(-5) M) exerted only a slight if any contractile effect. After the complete relaxation induced by sodium nitroprusside or SIN-1, the K+-channel blockers, tetraethylammonium, apamin and charybdotoxin, as well as the Ca2+ ionophore, A 23187, induced high-amplitude contractions, suggesting that the Ca2+ sensitivity of the contractile myofilaments was not affected. The results suggest that NO, released from NO donors increases the sarcoplasmic reticulum Ca2+ uptake thereby enhancing the vectorial sarcoplasmic reticulum Ca2+ release toward the plasmalemma to elicit membrane hyperpolarization and relaxation in guinea pig gastric fundus.

Cyclopiazonic acid-induced changes in contractile activity of smooth muscle strips isolated from cat and guinea-pig stomach.

The effects of cyclopiazonic acid (CPA), a specific inhibitor of sarcoplasmic reticulum Ca(2+)-ATPase, on contractile activity of circular smooth muscle strips isolated from the antrum, corpus and fundus regions of the cat and guinea-pig stomach were studied. Contractile activity was recorded under isometric conditions, in organ baths. CPA, concentration dependently (3 x 10(-7)-3 x 10(-5) M) increased the tone of the cat and guinea-pig gastric fundus and corpus as well as the amplitude of the phasic contractions of the cat corpus and antrum, affecting their frequency. CPA had a dual action on the phasic contractions of the guinea-pig antrum: an increase at low concentrations (up to 10(-6) M) and inhibition at high concentrations (10(-6)-3 x 10(-5) M). Tetrodotoxin (10(-6) M), atropine (10(-6) M) and N omega-nitro-L-arginine (10(-4) M) did not change significantly the effects of CPA. Nifedipine completely inhibited the CPA-induced phasic contractions and partly inhibited the CPA-induced tonic contractions. The nitric oxide-releasing agents, sodium nitroprusside (10(-3) M) and 3-morpholino-sydnonimine (10(-3) M), completely inhibited the CPA-induced tonic and phasic contractions. CPA induced tonic contractions in the cat and guinea-pig gastric fundus precontracted by acetylcholine (10(-5) M) and inhibited the acetylcholine (10(-6) M)-induced phasic contractions in the guinea-pig gastric antrum and corpus. The results suggest multiple roles for sarcoplasmic reticulum Ca2+ stores and sarcoplasmic reticulum Ca(2+)-ATPase in the shaping of spontaneous and evoked tonic and phasic contractions of the stomach, and highlight important species and tissue differences.

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus.

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca2+-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca2+-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca2+. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca2+-activated K+ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca2+ influx was triggered through the Na+/Ca2+ exchanger and the other Ca2+ entry pathways were blocked. Thapsigargin, another specific SR Ca2+-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca2+-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca2+ influx presumably via nifedipine-sensitive Ca2+ channels. This study suggests the important role of SR Ca2+-ATPase in the modulation of the muscle tone and the function of SR as a "buffer barrier" to Ca2+ entry in the cat gastric fundus smooth muscle.

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.