Muscle Of Guinea-pig Vas Deferens Research Articles

Protein kinase C-dependent inhibition of K+ currents in noradrenaline-induced depolarization of smooth muscle of guinea-pig vas deferens

Ionic mechanisms and signal transduction underlying noradrenaline (NA)-induced depolarization in single smooth muscle cells of guinea-pig vas deferens were studied. NA caused depolarization followed by action potentials through activation of α1-adrenoceptors. In the presence of nifedipine, no action potential was generated, and the magnitude of the depolarization depended on the concentration of NA (0.1-100 μm). NA, through α1-adrenoceptor activation, reduced the magnitude of membrane currents in response to voltage ramp pulses from -90 to -30 mV in a concentration-dependent manner. The reversal potential of the current inhibited by NA changed proportionally to the change in the equilibrium potential of K+, suggesting that NA inhibited K+ channel activity. Treatment of cells with GDPβS, an inhibitor of G proteins, or bisindolylmaleimide (BIM), a selective protein kinase C (PKC) inhibitor, prevented the NA inhibition of the currents. Application of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, mimicked the effect of NA. It is suggested that in the smooth muscle of guinea-pig vas deferens, activation of α1-adrenoceptors and the subsequent activation of PKC led to inhibition of K+ currents, which is responsible for the depolarization induced by NA.

Protein Kinase C‐Dependent Inhibition of K+ Currents in Noradrenaline‐Induced Depolarization of Smooth Muscle of Guinea‐Pig vas Deferens

Ionic mechanisms and signal transduction underlying noradrenaline (NA)-induced depolarization in single smooth muscle cells of guinea-pig vas deferens were studied. NA caused depolarization followed by action potentials through activation of 1-adrenoceptors. In the presence of nifedipine, no action potential was generated, and the magnitude of the depolarization depended on the concentration of NA (0.1-100 micrometer). NA, through 1-adrenoceptor activation, reduced the magnitude of membrane currents in response to voltage ramp pulses from -90 to -30 mV in a concentration-dependent manner. The reversal potential of the current inhibited by NA changed proportionally to the change in the equilibrium potential of K+, suggesting that NA inhibited K+ channel activity. Treatment of cells with GDPS, an inhibitor of G proteins, or bisindolylmaleimide (BIM), a selective protein kinase C (PKC) inhibitor, prevented the NA inhibition of the currents. Application of 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, mimicked the effect of NA. It is suggested that in the smooth muscle of guinea-pig vas deferens, activation of 1-adrenoceptors and the subsequent activation of PKC led to inhibition of K+ currents, which is responsible for the depolarization induced by NA.

Excitatory and inhibitory actions of norepinephrine on the Ba2+ current through L-type Ca2+ channels of smooth muscle cells of guinea-pig vas deferens.

The effect of norepinephrine (NE) was examined on the whole-cell Ba2+ current through L-type Ca2+ channels of freshly isolated smooth muscle cells of guinea-pig vas deferens. The magnitude of maximum Ba2+ current [1Ba(max)] varied in different cells, although the capacitance of the cell membrane was similar (approximately 50 pF). Application of dbcAMP augmented 1Ba(max) by 37%, which was canceled by Rp-cAMPs, while PMA decreased the current by 32%, which was canceled by staurosporine. NE increased 1Ba(max) of the cells which originally showed relatively small 1Ba(max), and decreased the current of the cells which showed larger 1Ba(max). In the presence of phentolamine, NE increased 1Ba(max), and this effect was remarkable in cells showed smaller 1Ba(max). In the presence of propranolol, NE decreased 1Ba(max). The excitatory beta-adrenoceptor activation was canceled by Rp-cAMPs, and the inhibitory alpha-adrenoceptor effect was canceled by staurosporine. It is suggested that NE shows dual (excitatory and inhibitory) actions on the L-type Ca2+ channels of smooth muscle of guinea-pig vas deferens. The excitatory beta-adrenoceptor action mediated through cAMP/PKA is predominant in cells with lower density of the Ca2+ channels, while inhibitory alpha-adrenoceptor action mediated through PKC is predominant in cells with higher channel density.

O-316 - Manganese-dependent norepinephrine-induced contractions in β-escin permeabilized smooth muscle of guinea-pig vas deferens.

O-316 - Manganese-dependent norepinephrine-induced contractions in β-escin permeabilized smooth muscle of guinea-pig vas deferens.

Evidence that ATP, released from the postsynaptic site by noradrenaline, is involved in mechanical responses of guinea-pig vas deferens: Cascade transmission

The release of endogenous ATP and [ 3H]noradrenaline and the mechanical response of the guinea-pig vas deferens to field stimulation of its motor nerves were examined using a perfusion system. The release of ATP at rest was 0.83 ± 0.13pmol/g per min and ATP released by field stimulation (8 Hz, 480 shocks) was 5.47 ± 1.23pmol/g. The evoked release was completely inhibited when Ca 2+ was removed and 1 mM EGTA was added, or by 1 μM tetrodotoxin. The release of ATP and [ 3H]noradrenaline in response to field stimulation was constant with an S 2/S 1 ratio of 1.10 ± 0.11 for ATP and 0.92 ± 0.03 for [ 3H]noradrenaline, respectively (where S 1 and S 2 are stimulation periods). Prazosin (1 μM), a potent α 1-adrenoceptor antagonist, significantly reduced the stimulation-evoked release of ATP by 75% and significantly reduced both mechanical twitch and tonic responses, but enhanced the release of [ 3H]noradrenaline. This finding indicates that there is an α 1-adrenoceptor-mediated release of endogenous ATP. However, the prazosin-insensitive portion of ATP release (25%) is considered to be of presynaptic origin. The stimulation of α 1-adrenoceptors by 1-noradrenaline or methoxamine in concentrations ranging from 10 to 100 μM resulted in a concentration-dependent release of ATP and a biphasic contraction of the vas deferens: a twitch response was followed by a tonic contraction. Prazosin (1 μM) completely prevented the effect of 1-noradrenaline or methoxamine on both ATP release and mechanical response. When Ca 2+ was omitted and EGTA (1 mM) was added, 1-noradrenaline was still able to release ATP but failed to produce contraction. Nifedipine, a Ca-channel and ATP receptor antagonist, reduced the twitch contraction and enhanced the release of ATP from muscle in response to noradrenaline administration. This finding indicates that the release of ATP from the muscle is not linked to mechanical contraction. When the vas deferens was made deficient in noradrenaline by 6-hydroxydopamine pretreatment (100 + 250rmmg/kg, i.p.), electrical field stimulation failed to release [ 3H]noradrenaline and ATP. Under these conditions, exogenous 1-noradrenaline was much more effective in releasing ATP from the smooth muscle and producing twitch responses, followed by a tonic contraction. After reserpine pretreatment (2 × 5mg/kg, i.p.), the field stimulation-evoked release of ATP and both phases of contraction were markedly reduced. These findings provide neurochemical evidence, from measurement of the release of ATP by the luciferin-luciferase method, that ATP is released from the smooth muscle of guinea-pig vas deferens in response to α 1-adrenoceptor stimulation by noradrenaline released from the sympathetic axon terminals. ATP released from the postsynaptic site in response to a chemical signal of presynaptic origin (noradrenaline) acts in loco via stimulation of P 2x receptors present on the smooth muscle outer surface. It produces a twitch response and contributes to the tonic contraction produced by noradrenaline. This leads to the proposal of a new type of transmission in smooth muscle—cascade transmission—where a transmitter, noradrenaline releases a secondary transmitter, ATP, from the postsynaptic site. This ATP, in addition to the ATP of presynaptic origin, becomes involved in the signal transmission via stimulation of postsynaptic P 2x receptors.

Two alpha-adrenergic contractions with different Ca2+ activation mechanisms in response to field nerve stimulation in the circular smooth muscle of guinea-pig vas deferens.

1. Two noradrenergic components of contraction were induced by field nerve stimulation of the circular smooth muscle of guinea-pig vas deferens. One component occurred during the stimulation (former-response) and the other component occurred after stimulation had ceased (after-response), with a distinct tension fall between these two responses. 2. The effects of stimulation frequency and duration upon these components were examined. 3. Prazosin (0.05-0.5 microM) suppressed both responses, whereas nifedipine (0.02 microM) suppressed only the after-response. 4. It is suggested that in this tissue there are two neurogenic alpha-adrenergic contractions which possess different Ca2+ activation mechanisms. The former-response is due to an increase in cytoplasmic Ca2+ concentration through a pathway independent of action potentials, while the after-response is attributed to action potentials arising as a secondary response to the released noradrenaline.

Pharmacological studies on the role of cholinergic nerves in the neuromuscular transmission in the circular smooth muscle of guinea-pig vas deferens.

Effects of various drugs were examined on the mechanical responses of the circular smooth muscle of guinea-pig vas deferens evoked by field stimulation and ACh. During field stimulation (0.1 msec, 3-80 Hz) a contraction consisting of two (initial and second) phases occurred. In addition, another contraction (after-response) occurred after the cessation of the stimulation particularly at higher frequencies (40-80 Hz). Guanethidine (2-20 microM) suppressed both the initial and second phases of the field stimulation-evoked responses. Prazosin (0.1-1 microM) suppressed the second phase but minimally affected the initial phase. Both phases were suppressed by atropine (0.1-1 microM) and potentiated by physostigmine (5 microM). dTC (0.5-20 microM) did not suppress either phase but potentiated the second phase. ACh (0.28-280 microM) produced a contraction, which was reduced by atropine but was little affected by either dTC or TTX. These results suggest that at the postsynaptic site of the neuromuscular junction in this tissue the sympathetic process activates the smooth muscle through two different types of receptor; one is non-noradrenergic and the other is an alpha-adrenoreceptor. In addition, a cholinergic process also activates the smooth muscle through muscarinic receptors.

Dual excitatory actions of acetylcholine at the neuromuscular junction in the guinea-pig vas deferens.

The action of acetylcholine (ACh) on the smooth muscle of guinea-pig vas deferens was studied using the sucrose-gap method. ACh, when applied at a concentration of 10(-6) M, evoked a depolarization of the smooth muscle membrane which was slow in time course (slow depolarization). When ACh was applied at higher concentrations, another depolarization which was fast in time course (fast depolarization) occurred, overlapping the early part of the slow depolarization. The magnitudes of both depolarizations were concentration-dependent on ACh. TTX and adrenergic receptor antagonists had little effect on either depolarizations, while guanethidine and nicotinic receptor antagonists mainly suppressed the fast depolarization. In contrast, atropine suppressed the slow depolarization. The membrane conductance observed by current application, was reduced during the slow depolarization, and the reversal potential of the depolarization was 18.3 mV negative to the resting membrane potential. Whereas, the reversal potential of the fast depolarization was 27.6 mV positive to the resting membrane potential. This reversal potential was quite similar to that of the adenosine triphosphate (ATP)-induced depolarization, previously observed in the same tissue. From these observations, it is suggested that in the guinea-pig vas deferens, ACh acts on nicotinic receptors at the sympathetic postganglionic nerve terminal, causing the release mostly of a non-adrenergic transmitter, probably ATP. In addition, ACh also acts on muscarinic receptors on the smooth muscle membrane, inducing membrane depolarization resulting from a reduction of the membrane conductance to potassium ions.

Evidence for suppression of potassium conductance by noradrenaline in smooth muscle of guinea-pig vas deferens.

The ionic dependency was examined in the reversal potential of the NA-induced membrane depolarization of smooth muscle of the guinea-pig vas deferens, using the double sucrose-gap method. In the control (5.9 mM K) solution and solutions containing 20 and 60 mM K ions, replaced with equimolar amount of Na ions, the reversal potential was negative to the membrane potential at the steady state in each solution, by 19.5, 15.1 and 13.2 mV, respectively. Taking account that the higher external K concentration causes the larger membrane depolarization, these results indicate that the reversal potential shifted toward the positive direction as the external K concentration was increased. On the other hand, the calculated equilibrium potential of K ions in those solutions is -88, -52 and -24 mV, respectively. In those solutions, furthermore, the equilibrium potential of Na ions is 42, 38 and 29 mV, respectively. However, the equilibrium potentials of Ca and Cl ions should be unchanged. Therefore, it is suggested that the reversal potential of the NA-induced depolarization varied in the given different solutions compatibly only with the change in the K equilibrium potential. This provides evidence that in this tissue, NA induces a reduction in the K conductance of the cell membrane.

Decrease in membrane conductance induced by noradrenaline in the smooth muscle of guinea-pig vas deferens.

The electrical response of the smooth muscle of guinea-pig vas deferens to exogenously applied noradrenaline (NA) was examined using the double sucrose-gap method. NA evoked a depolarization of the smooth muscle membrane which was associated with an increase in the size of electrotonic potentials. A conditioning depolarization of the membrane induced by current application enhanced the size of NA-induced depolarization, whereas a conditioning hyperpolarization reduced it. When a conditioning hyperpolarization of 25 mV in magnitude was applied, the direction of potential change induced by NA was reversed. These results are discussed with respect to the ionic mechanism of the electrical event in response to NA in this tissue.

Evidence for an increase in membrane conductance during adenosine triphosphate-induced depolarization in the guinea-pig vas deferens.

The effects of exogenously applied adenosine triphosphate (ATP) on the smooth muscle of guinea-pig vas deferens were studied with the double sucrose-gap method. ATP evoked a membrane depolarization which was associated with a decrease in the size of electrotonic potentials. Conditioning hyperpolarization induced by current application caused an increase in the magnitude of the ATP-induced depolarization; the larger the conditioning hyperpolarization, the greater the ATP-induced depolarization. These results are discussed with respect to the ionic mechanism of the electrical event in response to ATP in this tissue.

The effects of sodium nitroprusside on the phasic and tonic contraction of the potassium contracture of the smooth muscle of guinea-pig vas deferens and portal vein.

The effects of sodium nitroprusside on potassium-induced phasic and tonic contraction of the guinea pig vas deferens and portal vein were studied. Sodium nitroprusside at concentrations higher than 10(-4) M potentiated the phasic contraction of potassium contracture of the vas deferens, while it showed no or slight depressing effect on the tonic contraction. Sodium nitroprusside at the same concentration depressed both the phasic and tonic contractions of the portal vein. Sodium nitroprusside depolarized the membrane of the vas deferens but did not alter the depolarization by K-Tyrode's solution. Potentiation of the phasic contraction was also observed following the pretreatment with 20 mM K+. When sodium nitroprusside was applied during the course of the tonic contraction, marked relaxation was observed in the portal vein, while only slight relaxation was induced in the vas deferens even by high concentrations of the drug. Residual tension in the presence of sodium nitroprusside was completely abolished by the application of verapamil of the concentration higher than 10(-6) M. Relaxations of preparation by sodium nitroprusside were counteracted by increases in Ca concentration. When sodium nitroprusside was applied in the presence of moderately elevated-K+ (15 to 30 mM), an increase in tension was observed in the vas deferens and a decrease in tension was observed in the portal vein. The differences in the action of sodium nitroprusside on the vas deferens and on the portal vein may be explained by the differences in the action of the drug on cell membrane, especially on the membrane potential.

Requirements of external Ca and Na for the electrical and mechanical responses to noradrenaline in the smooth muscle of guinea-pig vas deferens.

Noradrenaline (NA) evoked a contraction which consisted of two components, an initial one associated with only depolarization and a second one with spike discharges. Both contractions were abolished in a Ca-free solution leaving only small depolarization. In a Na-free solution, the initial contraction was increased in keeping with the short term depolarization, while the second contraction was abolished. It is suggested that NA initially causes Ca influx which secondarily increases Na permeability.

Effect of cobalt on electrical and mechanical responses to exogenous noradrenaline and to field stimulations in the guinea-pig vas deferens.

Co2+ suppressed the mechanical responses evoked by field stimulations and by application of noradrenaline (NA) in the smooth muscle of guinea-pig vas deferens. However, the membrane excitation and force development evoked by depolarizing currents were not much affected in the Co2+ treatment. It is proposed that Co2+ suppresses NA induced contraction by inhibiting the development of the membrane depolarization.

Characteristics contractile response to the calcium ionophore, A23187, in guinea-pig vas deferens.

1. In the guinea-pig isolated vas deferens, the calcium ionophore, A23187, initially caused a phasic contraction followed by rhythmic activity. 2. Treatment with atropine, phentolamine, tetrodotoxin and reserpine modified neither of the components of the contraction induced by the ionophore. Verapamil and nifedipine abolished the rhythmic activity but had no effect on the phasic contraction. 4. In a calcium-free solution, both components of the contraction were abolished. 5. It is suggested that A23187 is able to cause contraction by a direct action on the smooth muscle of guinea-pig vas deferens.