Muscle Cells Of Guinea-pig Ileum Research Articles

O-224 - ATP-sensitive K+ channels in smooth muscle cells of guinea-pig ileum

O-224 - ATP-sensitive K+ channels in smooth muscle cells of guinea-pig ileum

Microtubule cytoskeleton involvement in muscarinic suppression of voltage-gated calcium channel current in guinea-pig ileal smooth muscle.

1. Effects of agents, which affect microtubule polymerization-depolymerization cycle, on Ba2+ current (IBa) flowing through voltage-gated Ca2+ channels and carbachol (CCh)-induced sustained suppression of IBa were examined in whole-cell voltage-clamped smooth muscle cells of guinea-pig ileum. 2. offchicine (100 microM) and vinblastine (100 microM), microtubule depolymerizers, increased the ampitude of IBa. Lumicolchicine (100 microM), an inactive analogue of colchicine, had no effect on IBa. 3. Taxol (1 - 100 microM), a microtubule polymerizer, decreased IBa in a concentration-dependent manner and accelerated the rate of inactivation of IBa. Baccatin III (100 microM), an inactive analogue of taxol, had no effect on IBa. 4. Colchicine (100 microM) and vinblastine (100 microM), but not lumicolchicine (100 microM), decreased or abolished the sustained component of CCh (10 microM)-induced IBa suppression. 5. Pretreatment with taxol (10 - 100 microM) resulted in a concentration-dependent decrease in IBa and the action of CCh on IBa. The inhibitory effects of taxol and CCh on IBa were not additive. 6. Colchicine (100 microM) or taxol (100 microM) had no effect on voltage-gated K+ channel current or CCh-induced non-selective cationic channel current. 7. These results suggest that polymerization of microtubules leads to suppression of Ca2+ channel activity, and that muscarinic sustained suppression of Ca2+ channel current is mediated by a signal transduction element which involves microtubule cytoskeleton.

Involvement of Ca2+ mobilizing mechanisms in muscarinic receptor-mediated depolarization in single smooth muscle cells of guinea-pig ileum.

Involvement of Ca2+ mobilizing mechanisms in muscarinic receptor-mediated depolarization in single smooth muscle cells of guinea-pig ileum.

Carbachol-induced oscillations in membrane potential and [Ca2+]i in guinea-pig ileal smooth muscle cells.

1. Cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential were simultaneously recorded from single smooth muscle cells of guinea-pig ileum, using a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques. 2. Carbachol (CCh, 2 microM) produced oscillatory changes in [Ca2+]i and membrane potential which coincided well in time with each other, and peaks of membrane potential oscillations reached a saturated level of around -7 mV. Thapsigargin (1 microM) abolished these effects of 2 microM CCh. La3+ (3 microM) immediately prevented the discharge of spike potentials, but allowed both on-going oscillatory responses to persist for a while. 3. CCh (0.25-0.75 microM) caused membrane potential and [Ca2+]i to oscillate in some 20 % of cells studied. Every membrane potential oscillation was preceded by the discharge of single or multiple spike potentials. The effects of CCh were readily abolished by La3+ (3 microM). 4. In cells exhibiting no oscillatory response to 0.25-0.75 microM CCh, an electrically evoked action potential usually generated changes in [Ca2+]i and membrane potential similar to those following spontaneously evoked action potentials, and sometimes it did so only after [Ca2+]i or InsP3 had been slightly elevated by repeatedly evoking action potentials or by increasing CCh concentration in the bath medium. 5. The results suggest that in ileal smooth muscle cells, the oscillations of [Ca2+]i and membrane potential arising from muscarinic stimulation result from release of Ca2+ from internal stores and that there is a Ca2+-induced potentiation of coincidently elicited cation channel openings. Under weak muscarinic stimulation, Ca2+ entry upon action potential discharge can trigger such a release of stored Ca2+, resulting in synchronous generation of a large rise in [Ca2+]i and a slow, large membrane depolarization.

Characterization of action potential-triggered [Ca2+]i transients in single smooth muscle cells of guinea-pig ileum.

1. To characterize increases in cytosolic free Ca2+ concentration ([Ca2+]i) associated with discharge of action potentials, membrane potential and [Ca2+]i were simultaneously recorded from single smooth muscle cells of guinea-pig ileum by use of a combination of nystatin-perforated patch clamp and fura-2 fluorimetry techniques. 2. A single action potential in response to a depolarizing current pulse elicited a transient rise in [Ca2+]i. When the duration of the current pulse was prolonged, action potentials were repeatedly discharged during the early period of the pulse duration with a progressive decrease in overshoot potential, upstroke rate and repolarization rate. However, such action potentials could each trigger [Ca2+]i transients with an almost constant amplitude. 3. Nicardipine (1 microM) and La3+ (10 microM), blockers of voltage-dependent Ca2+ channels (VDCCs), abolished both the action potential discharge and the [Ca2+]i transient. 4. Charybdotoxin (ChTX, 300 nM) and tetraethylammonium (TEA, 2 mM), blockers of large conductance Ca2+-activated K+ channels, decreased the rate of repolarization of action potentials but increased the amplitude of [Ca2+]i transients. 5. Thapsigargin (1 microM), an inhibitor of SR Ca2+-ATPase, slowed the falling phase and somewhat increased the amplitude, of action potential-triggered [Ca2+]i transients without affecting action potentials. In addition. in voltage-clamped cells, the drug had little effect on the voltage step-evoked Ca2+ current but exerted a similar effect on its concomitant rise in [Ca2+]i to that on the action potential-triggered [Ca2+]i transient. 6. Similar action potential-triggered [Ca2+]i transients were induced by brief exposures to high-K+ solution. They were not decreased, but rather increased, after depletion of intracellular Ca2+ stores by a combination of ryanodine (30 microM) and caffeine (10 mM) through an open-lock of Ca2+-induced Ca2+ release (CICR)-related channels. 7. The results show that action potentials, discharged repeatedly during the early period of a long membrane depolarization, undergo a progressive change in configuration but can each trigger a constant rise in [Ca2+]i. Intracellular Ca2+ stores have a role, especially in accelerating the falling phase of the action potential-triggered [Ca2+]i transients by replenishing cytosolic Ca2+. No evidence was provided for the involvement of CICR in the action potential-triggered [Ca2+]i transient.

The Calcitonin Gene-Related Peptide Activates Both cAMP and NO Pathways to Induce Relaxation of Circular Smooth Muscle Cells of Guinea-Pig Ileum

Rekik, M., M. Delvaux, J. Frexinos, and L. Bueno. Calcitonin gene-related peptide activates both cAMP and NO pathways to induce relaxation of circular smooth muscle cells of guinea-pig ileum. Peptides 18(10) 1517–1522, 1997.—The direct effects and the intracellular pathways of rCGRP were investigated on smooth muscle cells (SMC) isolated by enzymatic digestion from the circular and longitudinal layers of guinea-pig ileum. In circular SMC, rCGRP inhibited CCK8-induced contraction in a concentration-dependent manner (Cmax = 100 μ M and EC 50 = 0.7 ± 0.4 n M). Preincubation of SMC with 1 μ M Rp-cAMPs, a cAMP antagonist, abolished the relaxing effect of rCGRP; moreover, preincubation of SMC with 100 μ M L-NAME, an inhibitor of NOS, inhibited the relaxing effect of rCGRP. hCGRP(8-37), a selective antagonist of rCGRP receptors, inhibited the rCGRP-induced relaxation in a concentration dependent manner whereas the vasoactive intestinal polypeptide (VIP) antagonist had no significant effect. In longitudinal SMC, rCGRP-induced relaxation was abolished by Rp-cAMPs, whereas L-NAME had no effect. In conclusion, rCGRP triggers different intracellular pathways to induce relaxation of circular or longitudinal intestinal SMC; cAMP is involved in cells from both layers while nitric oxide (NO) is involved only in relaxation of circular SMC.

P-659 - Some properties of carbachol-induced oscillations in membrane potential in single smooth muscle cells of guinea-pig ileum

P-659 - Some properties of carbachol-induced oscillations in membrane potential in single smooth muscle cells of guinea-pig ileum

Carbachol-induced [Ca2+]i oscillations in single smooth muscle cells of guinea-pig ileum.

1. Changes in cytosolic Ca2+ concentration ([Ca2+]i) produced by carbachol (CCh) were measured in single smooth muscle cells of guinea-pig ileum using a Ca(2+)-sensitive fluorescent dye, fura-2, to clarify the underlying mechanisms of muscarinic [Ca2+]i oscillations. 2. Half of the cells, when exposed to 0.2 microM CCh, exhibited repeated changes in [Ca2+]i giving a serrated appearance. The oscillatory changes in [Ca2+]i were very similar to those evoked by increasing extracellular K(+) concentration ([K+]o) to 30 mM, which were abolished by removal of extracellular Ca2+, nifedipine and La3+, but remained unchanged after depletion of internal Ca2+ stores with cyclopiazonic acid, thapsigargin and ryanodine. 3. Every individual [Ca2+]i oscillation was just like a [Ca2+]i increase generated spontaneously in about 8% of cells or triggered by an action potential evoked by a current pulse in current-clamped cells. 4. In the remaining half of the cells exposed to 0.2 microM CCh, slower [Ca2+]i oscillations were elicited and every individual [Ca2+]i oscillation was always preceded by the fast brief increase in [Ca2+]i. 5. [Ca2+]i oscillations elicited by 2 microM CCh were temporally and functionally distinct from those induced by high [K+]o. They were more or less regular in the periodicity and pattern, comprised pacemaker potential-like [Ca2+]i increases or sinusoidal types of [Ca2+]i increases, and could be elicited even in 100 mM K+(o). 6. Removal of extracellular Ca2+ or application of nifedipine, methoxyverapamil (D600), diltiazem or La3+ during CCh (2 micro M)-induced [Ca2+]i oscillations caused them to disappear. In cells i which internal Ca2+ stores were depleted, 2 microM CCh did not evoke [Ca2+]i oscillations but occasionally induced single or repeated generation of the increase in [Ca2+]i with a serrated appearance. 7. The results indicate that CCh can induce two types of [Ca2+]i oscillation in guinea-pig ileal smooth muscle cells; one arises from Ca2+ influx associated with action potential discharges and the other from periodic release of Ca2+ from internal stores. The latter [Ca2+]i oscillation requires extracellular Ca2+ to sustain it.

P2-189 - Carbachol-induced [Ca2+]i oscillations in single smooth muscle cells of guinea-pig ileum

1. Changes in cytosolic Ca2+ concentration ([Ca2+]i) produced by carbachol (CCh) were measured in single smooth muscle cells of guinea-pig ileum using a Ca(2+)-sensitive fluorescent dye, fura-2, to clarify the underlying mechanisms of muscarinic [Ca2+]i oscillations. 2. Half of the cells, when exposed to 0.2 microM CCh, exhibited repeated changes in [Ca2+]i giving a serrated appearance. The oscillatory changes in [Ca2+]i were very similar to those evoked by increasing extracellular K(+) concentration ([K+]o) to 30 mM, which were abolished by removal of extracellular Ca2+, nifedipine and La3+, but remained unchanged after depletion of internal Ca2+ stores with cyclopiazonic acid, thapsigargin and ryanodine. 3. Every individual [Ca2+]i oscillation was just like a [Ca2+]i increase generated spontaneously in about 8% of cells or triggered by an action potential evoked by a current pulse in current-clamped cells. 4. In the remaining half of the cells exposed to 0.2 microM CCh, slower [Ca2+]i oscillations were elicited and every individual [Ca2+]i oscillation was always preceded by the fast brief increase in [Ca2+]i. 5. [Ca2+]i oscillations elicited by 2 microM CCh were temporally and functionally distinct from those induced by high [K+]o. They were more or less regular in the periodicity and pattern, comprised pacemaker potential-like [Ca2+]i increases or sinusoidal types of [Ca2+]i increases, and could be elicited even in 100 mM K+(o). 6. Removal of extracellular Ca2+ or application of nifedipine, methoxyverapamil (D600), diltiazem or La3+ during CCh (2 micro M)-induced [Ca2+]i oscillations caused them to disappear. In cells i which internal Ca2+ stores were depleted, 2 microM CCh did not evoke [Ca2+]i oscillations but occasionally induced single or repeated generation of the increase in [Ca2+]i with a serrated appearance. 7. The results indicate that CCh can induce two types of [Ca2+]i oscillation in guinea-pig ileal smooth muscle cells; one arises from Ca2+ influx associated with action potential discharges and the other from periodic release of Ca2+ from internal stores. The latter [Ca2+]i oscillation requires extracellular Ca2+ to sustain it.

P2-178 - Evidence against involvement of arachidonic acid in muscarinic suppression of voltage-gated Ca2+ channel current in smooth muscle cells of guinea-pig ileum

P2-178 - Evidence against involvement of arachidonic acid in muscarinic suppression of voltage-gated Ca2+ channel current in smooth muscle cells of guinea-pig ileum

Specific receptor for vasoactive intestinal contractor in myenteric cholinergic neurones

The receptor for vasoactive intestinal contractor (VIC) involved in acetylcholine release was examined in the guinea-pig ileum. VIC (10 −9 to 10 −7 M) evoked tritium outflow ileal strips preloaded with [ 3H]choline, in a concentration-dependent manner. The evoked tritium outflow was Ca 2+-dependent and tetrodotoxin-sensitive, suggesting that VIC acts on somato-dendritic regions of the cholinergic neurones to stimulate acetylcholine release. The response to VIC did not change after desensitization to either endothelin-1, endothelin-2 or endothelin-3, hence there is probably no cross-desensitization in evoked tritium outflow between VIC and endothelins. VIC-induced contractions consisted tetrodotoxin-sensitive and -resistant contractions, and the tetrodotoxin-resistant contraction was abolished after desensitization to either endothelin-1, endothelin-2 or endothelin-3. These results indicate that the VIC-specific receptor is located on cholinergic neurones, but not on the smooth muscle cells of guinea-pig ileum.

P-170 - Oscillatory current response to activation of muscarinic receptors in smooth muscle cells of guinea-pig ileum

P-170 - Oscillatory current response to activation of muscarinic receptors in smooth muscle cells of guinea-pig ileum

Effect of external Cd2+ and other divalent cations on carbachol‐activated non‐selective cation channels in guinea‐pig ileum.

1. Inhibitory actions of the external divalent cations, Cd2+, Ni2+, Mn2+ and Co2+ on carbachol (CCh)-induced inward current (Ins,ACh) were investigated in caesium aspartate-loaded single longitudinal smooth muscle cells of guinea-pig ileum, using a rapid solution-switching device, under voltage-clamped conditions. 2. Cd2+ (20-1000 microM), when added to the external solution, reduced Ins,ACh rapidly and reversibly. This effect occurred dose dependently, the relationship being adequately described by a Michaelis-Menten equation with a Hill coefficient (n) of 1.0 and a dissociation constant (Kd) of 98 microM. 3. The inhibitory action of Cd2+ was associated neither with agonist concentration (CCh) nor with changes in reversal potential, and was voltage independent. 4. The appearance and removal of the effect of Cd2+ were both rapid (a few hundred milliseconds), in sharp contrast with a relatively slow time course of atropine or CCh action (of the order of seconds). 5. Other divalent cations (Ni2+, Mn2+, Co2+, Mg2+) applied externally also suppressed Ins,ACh, but less potently. The sequence of apparent potency was Cd2+ (98 microM) greater than or equal to Ni2+ (131 microM) much greater than Co2+ (700 microM) greater than or equal to Mn2+ (1000 microM) much greater than Mg2+ (approximately 10 mM). 6. External Ca2+ increased Ins,ACh dose dependently and antagonized the inhibitory effect of Cd2+. However, this effect may not be a simple competition with Cd2+. 7. These results show that external divalent cations strongly modulate Ins,ACh channels, possibly through direct interaction with the channel protein.

Pharmacological evidence for a spasmogenic agent inDatura root extract tested on dispersed smooth muscle cells of guinea-pig ileum

The effect of acetylcholine, scopolamine, and Datura leaf and root extracts on the contractility of dispersed muscle cells of guinea-pig ileum was studied. Scopolamine and crude Datura leaf extract alone or together had no significant effect on contraction of smooth muscle cells. Acetylcholine and Datura root extract either alone or together caused a significant (30%) contraction. Datura root extract reversed the effect of both scopolamine and Datura leaf extract suggesting the presence of an unknown spasmogenic factor in Datura root extract.

Action of morphine on the neuro-effector transmission in the guinea-pig ileum and in the mouse vas deferens.

Effects of morphine on the neuro-effector junction of the guinea-pig ileum or mouse vas deferens were investigated by the micro-electrode and double sucrose gap methods. 1. Morphine (10(-8)-10(-5) M) did not change the membrane potential, membrane resistance and electrical threshold required to produce the action potential of smooth muscle cells of guinea-pig ileum or mouse vas deferens. 2. Morphine (10(-8)-10(-7) M) markedly suppressed the amplitude of excitatory junction potential (e.j.p.) of ileum or that of vas deferens. However the same concentration of morphine did not suppress the inhibitory junction potential recorded from the guinea-pig ileum or the facilitation phenomena observed with repetitive stimulation in the mouse vas deferens. In addition, this opiate (3.5 x 10(-5) M) did not alter the amplitude or the frequency of miniature excitatory junction potential recorded from the mouse vas deferens. 3. Naloxone (3.5 x 10(-7) M), itself, exerted no effect on the membrane potential and amplitude of the e.j.p. After pretreatment with naloxone, however, the inhibitory action of morphine on the e.j.p. was suppressed. 4. The extracellularly recorded action potential from the small nerve bundle innervating the mouse vas deferens was not affected by morphine (3.5 x 10(-5) M). 5. The amplitude of the e.j.p. of the guinea-pig ileum was dependent on the concentration of [Ca]o. When [Ca]o and the relative amplitude of e.j.p. were plotted on a double logarithmic scale, the above relation yielded a straight line with a slope of 1.7. Application of morphine resulted in a reduction in the slope of the straight line to 1.0. 6. These results indicate that morphine probably suppresses the influx of Ca during spike electrogenesis in the nerve terminal, however, there is no modification of the action of Ca in nerve terminals.