A Denhertog

The effect of apamin on the smooth muscle cells of the guinea-pig taenia coli

The polypeptide apamin caused a small depolarization of the muscle cell membrane of the guinea-pig taenia coli accompanied by enhancement of spike activity and a concomitant muscle contration. The membrane hyperpolarization evoked by intramural stimulation of the non-adrenergic inhibitory nerves (inhibitory junction potential) was reduced by apamin; the antagonism being non-competitive in nature. The rebound depolarization and contraction following the inhibitory junction potential was enhanced by apamin. The membrane hyperpolarization induced by the purinergic compound ATP and by the sympathomimetic adrenaline was converted to a depolarization in the presence of apamin. This depolarization resulted in an increased spike activity and muscle contraction. This was followed by membrane hyperpolarization and muscle relaxation after washout of the drugs. These findings indicate that apamin is a non-competitive, non-specific antagonist of the non-adrenergic inhibitory transmitter and that the inhibitory junction potential and the rebound are mutually independent phenomena.

The action of apamin on guinea-pig taenia caeci

Apamin (10(-7) M), a substance extracted from bee venom (apis mellifica) causes stimulation of the taenia caeci as seen from an increase in spike activity. The inhibitory effect of ATP or adrenaline (Adr) was reflected by hyperpolarization of the muscle cell, cessation of spike activity and relaxation of the muscle. The 42K efflux and the membrane conductance were enhanced in the presence of these substances. Apamin converted the hyperpolarization caused by ATP or Adr into a transient depolarization which produced contraction of the muscle cells. The changes in membrane conductance and 42K efflux were diminished by the bee toxin. Furthermore, the potassium-dependent phase of the action potential was lengthened by apamin. Reduction of the extracellular chloride or sodium concentration, blockade of the nervous system by TTX (3 x 10(-7) M) or inhibition of spike activity by D600 (3 x 19(-6) M) did not affect the excitatory and blocking action of apamin. A high concentration of the calcium antagonist D600 (10(-4) M) or omission of extracellular calcium was needed to reduce the transient depolarization evoked by ATP or Adr in the presence of apamin. It is concluded that apamin prevents the opening of the ATP- and Adr-sensitive and voltage-dependent potassium channels in guinea-pig taenia caeci.

The inhibitory action of suramin on the P2-purinoceptor response in smooth muscle cells of guinea-pig taenia caeci

The effect of suramin on the smooth muscle cell response of guinea-pig taenia caeci to P2-purinoceptor and alpha 1-adrenoceptor stimulation was measured. The ATP-induced relaxation in potassium (20 mM) pre-contracted taenia caeci was inhibited by suramin (3 x 10(-4) M). The P2-purinoceptor-induced hyperpolarization elicited by ATP both in the presence and absence of calcium was also reduced by suramin. The alpha 1-adrenoceptor-mediated relaxation evoked by phenylephrine was only affected by suramin at low concentrations of the agonist. The results indicate that suramin inhibits the ATP response by interacting with P2y-purinoceptors.

P2-purinoceptor-activated membrane currents and inositol tetrakisphosphate formation are blocked by suramin

The effect of suramin on the ATP-induced response in vas deferens DDT1 MF-2 smooth muscle cells was studied. Stimulation of P2-purinoceptors by ATP caused a change in membrane currents, measured by using the whole-cell patch-clamp configuration, and enhanced the formation of inositol phosphates, as analysed by high performance liquid chromatography. The ATP-induced membrane current consisted of a triphasic response, carried by a fast inward current, followed by a transient outward current and a sustained inward current. Inositol tetrakisphosphate (InsP4) formation increased in the presence of ATP. The formation of the isomers Ins(1,3,4,5)P4, Ins(1,3,4,6)P4 and Ins(3,4,5,6)P4 increased significantly after 5 min stimulation with ATP. Suramin inhibited the ATP-evoked membrane currents and the ATP-induced formation of inositol tetrakisphosphate isomers concentration dependently, but did not affect the basal inositol phosphate levels in the absence of ATP. These results indicate that suramin inhibits ATP-activated cellular processes in DDT1 MF-2 vas deferens cells, most likely by acting on P2-purinoceptors.

Suramin reverses non-depolarizing neuromuscular blockade in rat diaphragm

Unexpectedly, it was observed that the P2-purinoceptor antagonist, suramin (10 microM to 1 mM), reversed the muscle paralysis caused by structurally unrelated non-depolarizing relaxants. Suramin competitively reversed the blocking action of pancuronium. Both the pre- and postsynaptic blockade of nicotinic receptors by pancuronium was counteracted, as shown by the action of suramin, using train-of-four stimulation. Suramin did not affect the paralysis caused by the depolarizing relaxant, succinylcholine. The reversal action of suramin was not due to an increase in the acetylcholine concentration in the synaptic cleft, since neither the contraction of preparations partially paralysed by diminished acetylcholine release in the presence of low Ca2+ or high Mg2+ nor acetylcholinesterase activity were affected. Suramin did not affect the reduction in twitch tension caused by adenosine and potentiated the ATP-induced reduction in twitch, indicating that ATP-sensitive receptors are not involved in the reversal action of suramin. Consequently, these results suggest that the action of suramin is due to binding with a site on the acetylcholine receptor also occupied by non-depolarizing relaxants, but different from the site occupied by succinylcholine.

INOSITOL PHOSPHATES FORMED IN RAT AORTA AFTER ALPHA-1-ADRENOCEPTOR STIMULATION ARE INHIBITED BY FORSKOLIN

Rat aortic smooth muscle rings without endothelial cells were subjected to alpha 1-adrenoceptor stimulation. We measured the contractile state of the smooth muscle cells and the formation of inositol phosphates (InsPs) on receptor stimulation. Using different extracellular calcium-containing solutions (2.5 mM, 0.1 mM and Ca(2+)-free) enabled us to discriminate three contractile phases after noradrenaline (10(-5) M) stimulation: an initial fast contraction (15 s) and a fast and slow component of the sustained contraction, which was established 10 min after stimulation. Under normal calcium conditions in the presence of 10 mM LiCl the formation of Ins(1,4,5)P3 was increased predominantly after stimulation, while the formation of Ins(1,3,4)P3, Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and InsP5/InsP6 was also stimulated. The cAMP-inducing agent forskolin (0.5 microM) induced a relaxation of the basal tone and increased the level of the InsP4 isomers. The noradrenaline-induced contractile responses as well as the formation of InsP fractions mentioned were inhibited by forskolin. Further an increase in the formation of phosphatidylinositol bisphosphate was observed. It is concluded that in rat aorta InsPs and in particular Ins(1,4,5)P3 is involved in the different contractile phases caused by alpha 1-adrenoceptor stimulation. The relaxation induced by forskolin under these circumstances could be explained by an interaction of forskolin, most likely via the formation of cAMP, with InsPs formation at the level of phospholipase C activation.

The contribution of calcium and potassium to the α-action of adrenaline on smooth muscle cells of the portal vein, pulmonary artery and taenia caeci of the guinea-pig

The role of calcium and potassium in the alpha-action of adrenaline in pulmonary artery and portal vein was compared with that in taenia caeci by measuring changes in membrane potential, muscle contraction and ion fluxes in quiescent preparations from guinea-pigs (23 degrees C). The depolarization evoked by adrenaline (5 x 10(-8)-3 x 10(-5) M) was sustained in portal vein; in pulmonary artery it declined to a constant level after reaching an initial maximum. In calcium-free medium (20 min) containing EGTA (0.4 mM) and high magnesium (6.2 mM) adrenaline did not affect the membrane potential or the contractile state of the portal vein. Under these conditions the sustained phase of the response was abolished in the pulmonary artery; the remaining transient depolarization and contraction could be evoked only once. Adrenaline (3 x 10(-5) M) caused an increased 45Ca loss and 86Rb loss from the pulmonary artery and taenia caeci in calcium-free solution; a second addition of adrenaline to the calcium-free solution did not enhance the 45Ca loss from these tissues. The portal vein responded with an enhanced 86Rb loss on addition of the alpha-agonist. The bee toxin apamin (3 x 10(7) M) did not modify the depolarization, the contraction or the 45Ca and 86Rb fluxes evoked by adrenaline in the blood vessels. Enhancement of the 86Rb loss from taenia in the presence of adrenaline was prevented by apamin, but the excess loss of 45Ca was not abolished. It is concluded that adrenaline enhances cytoplasmic calcium by promoting calcium entry from the extracellular space in portal vein. In pulmonary artery and taenia caeci this is accompanied by mobilization of calcium from a cellular structure. Calcium entry facilitates triggering of the contractile proteins in vascular smooth muscle and is associated with membrane depolarization; in taenia caeci the mobilization of calcium caused by alpha-receptor activation is associated with the opening of potassium channels producing hyperpolarization and accordingly relaxation of the smooth muscle cells.

The action of prostaglandin E2 on the smooth muscle cell of the guinea-pig taenia coli

Abstract The effect of prostaglandin E 2 (PGE 2 ) on the smooth muscle cell of guinea pig taenia coli was investigated by measuring changes in membrane potential, membrane resistance, muscle contraction and ion fluxes. The action of PGE 2 caused depolarization of the cell membrane, and increased spike activity and muscle contraction. The amplitude of the PGE 2 depolarization was concentration-dependent. The action of PGE 2 could be mimicked by depolarizing the muscle membrane with an external current with as result a decrease in membrane resistance similar to that observed in the presence of PGE 2 . The PGE 2 depolarization was not abolished when the sodium pump was inhibited by omitting external potassium or in the presence of K-strophantin, persisted in low chloride Krebs and in low sodium solution, but was reduced in calcium-free solution and in the presence of D600. 45 Ca influx was enhanced by PGE 2 , but 45 Ca efflux was not affected. The 42 K efflux was increased in the presence of PGE 2 , but this response was blocked by Ca-free solution. These results indicate that calcium permeability is enhanced by PGE 2 , and that this enhancement is associated with a depolarization of the muscle cell membrane of the guinea-pig taenia coli.

P2 –Purinoceptor-mediated inositol phosphate formation in relation to cytoplasmic calcium in DDT1 MF-2 smooth muscle cells

The effect of P2 purinoceptor stimulation on inositol phosphate (InsP) formation in relation to the intracellular Ca2+ concentration was measured in vas deferens DDT1 MF-2 smooth muscle cells. The different [3H]myo-inositol-labelled InsP fractions were analyzed by high performance liquid chromatography and intracellular Ca2+ was determined by measuring fluorescence using Indo-1 as indicator. Stimulation with ATP (10(-4) M) resulted in an enhanced formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP1, InsP2, InsP3 and InsP4), but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). The putative second messenger Ins(1,3,4,5)P4 rapidly increased after addition of the agonist, reaching a maximum after about 2 min. The isomer Ins(1,4,5)P3 showed a delayed rise starting after about 2 min. The formation of Ins(1,3,4,5)P4 in the presence of ATP (2 min) was concentration-dependent, reaching a half maximal value at about 50 microM of the agonist. The intracellular Ca2+ concentration showed an initial increase after P2 purinoceptor stimulation, reaching a plateau after 2 min. Both the top of the initial phase and the plateau value of the response reached a half maximal value at an ATP concentration of about 7 microM. This Ca2+ response could be evoked repeatedly by ATP and was not affected by diltiazem (10(-5) M). In the absence of external Ca2+, the internal Ca2+ concentration increased transiently in the presence of ATP without showing the plateau phase. This response could be evoked only once under Ca2(+)-free conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Modification of α1-receptor-operated channels by mebeverine in smooth muscle cells of guinea-pig taenia caeci

Abstract Changes in the potential and contractility of smooth muscle cells of guinea-pig taenia caeci were measured (22°C) in order to investigate the effect of mebeverine, a derivative of β-phenylethylamine, on α 1 -receptor-operated ion channels in particular. Mebeverine (6×10 −6 M showed atropine-like properties by shifting to the right the concentration-response curve obtained with carbachol. Hyperpolarization and cessation of spike activity of the muscle cells, accompanied by an increase amplitude of the electronic potential were observed in the presence of mebeverine (6 × 10 −5 ) after block of the α 2 -, β- and muscarinic receptors. This effect of mebeverine was not observed in low-sodium solution (23.8 mM), suggesting that mebeverine decreased sodium permeability. The α 1 -receptor-induced hyperpolarization caused by adrenaline (3 × 10 −6 M) in the presence of mebeverine declined after reaching an initial maximum. The hyperpolarization induced by a second addition of adrenaline to the preparation was decreased and sustained in the presence of mebeverine, while the decrease of the electronic potential evoked during the α 1 response was less pronounced. The transient hyperpolarization representing the α 1 response in the absence of extracellular calcium developed more slowly in the presence of mebeverine, the area of the response being constant. When the experiment was continued in calcium-free solution after a short exposure to calcium-containing Krebs solution still in the presence of mebeverine, the α 1 -receptor-induced hyperpolarization was suppressed. Depolarization of the muscle cells was observed on addition of adrenaline to the preparation in calcium-free conditions and in the presence of apamin (3 × 10 −7 M). Mebeverine affected this reversed α 1 response similarly to the hyperpolarization observed without apamin. The results show that mebeverine inhibited the α 1 action by limiting the amount of available calcium mobilized on receptor stimulation of the smooth muscle cells of guinea-pig taenia caeci.