Presence Of Apamin Research Articles

BK1 and BK2 bradykinin receptors in the rat duodenum smooth muscle.

1. The dual action of bradykinin (relaxation and contraction) on the rat duodenum was investigated by studying its effect on adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in cultured duodenal smooth muscle cells, and the effects of apamin on the isolated muscle responses to agonists and antagonists of BK1 and BK2 receptors. 2. No change was observed in the cyclic AMP content of cultured cells incubated with up to 100 nM bradykinin. 3. Apamin (100-500 nM) inhibited the relaxant component and enhanced the contractile component of the responses to bradykinin and to the BK2-specific analogue [Thi5,8,D-Phe7]-bradykinin. 4. Apamin (100-500 nM) did not affect the contractile response of stretched duodenum preparation to the BK1-specific agonist des-Arg9-bradykinin. 5. The BK2 antagonist, [D-Arg0Hyp3Thi5,8,D-Phe7]-bradykinin, at a concentration which completely inhibited the relaxant response to bradykinin and to [Thi5,8,D-Phe7]-bradykinin, also prevented the contraction in response to either agonist in the presence of apamin. 6. Our results demonstrate two populations of bradykinin receptors in rat duodenum: a BK2 subtype responsible for the biphasic response of the non-stretched duodenum, and a BK1 subtype responsible for the contractile effect on the stretched tissue.

Tachykininergic transmission to the circular muscle of the guinea-pig ileum: evidence for the involvement of NK2 receptors.

1. The effect of newly developed, receptor-selective tachykinin antagonists (GR 71,251 for NK1 receptors, MEN 10,376 and L 659,877 for NK2 receptors) on noncholinergic transmission to the circular muscle of the guinea-pig ileum has been investigated. 2. In circular muscle strips of the ileum, electrical field stimulation in the presence of atropine (2 microM) and apamin (0.1 microM) evoked a complex motor response. The tonic primary contraction in this response was reduced by GR 71,251 (10 microM) and MEN 10,376 (3-10 microM) but not by L 659,877 (up to 10 microM). The presence of apamin was necessary in this experimental arrangement to unmask an atropine-resistant primary contraction, sensitive to tachykinin antagonists. The motor response was abolished by tetrodotoxin. 3. In circular strips of the ileum GR 71,251 (10 microM) inhibited the tonic contraction produced by [Sar9] substance P sulphone, a selective NK1 receptor agonist but not that produced by [beta Ala8] neurokinin A (4-10), a selective NK2 receptor agonist. By contrast, MEN 10,376 antagonized the effect of the NK2 agonist while leaving the response to the NK1 agonist unaffected. 4. In whole segments of the ileum, distension of the gut wall by an intraluminal balloon placed at about 1 cm from the point of recording of mechanical activity of the circular muscle produced atropine-sensitive phasic contractions (ascending enteric reflex). In the presence of atropine (2 microM), a noncholinergic response was elicited, which required larger volumes of distension that the cholinergic one. The atropine-resistant ascending enteric reflex was enhanced by apamin (0.1 microM) and abolished by tetrodotoxin, either in the presence or absence of apamin.5. MEN 10,376 (3-lOmicroM) inhibited the atropine-resistant ascending enteric reflex in the presence of apamin while GR 71,251 or L 659,877 (10 microM each) were ineffective. MEN 10,376 inhibited the atropine-resistant ascending enteric reflex to a larger extent in the absence than in the presence of apamin and also slightly inhibited the ascending enteric reflex in the absence of atropine.6. These findings provide evidence for an involvement of NK2 tachykinin receptors in excitatory transmission to the circular muscle of the guinea-pig ileum. NK2 receptors are also involved in the physiological-like circular muscle activation produced by stimulation of intramural neuronal pathways which subserve the atropine-resistant ascending enteric reflex.

Both ATP and the peptide VIP are inhibitory neurotransmitters in guinea-pig ileum circular muscle.

1. Intracellular membrane potential recordings were made from circular smooth muscle cells of the guinea-pig ileum in the presence of atropine (1 microM) and nifedipine (0.1 microM) at 30 degrees C. 2. Perfusion with adenosine triphospate (ATP, 100 microM) and vasoactive intestinal peptide (VIP, 2 microM) resulted in membrane hyperpolarizations of 6.4 +/- 0.3 and 6.8 +/- 0.3 mV, respectively. Picospritzes of ATP (10 mM in pipette) and VIP (100 microM in pipette) resulted in membrane hyperpolarizations of 6.9 +/- 0.4 and 6.3 +/- 0.4 mV, respectively. 3. The ATP-induced hyperpolarizations were antagonized by alpha, beta-methylene ATP desensitization (100 microM for 30 min) and the ATP antagonist Reactive Blue 2 (200 microM), but were unaffected by the VIP antagonist VIP 10-28 (1 microM). 4. The VIP-induced hyperpolarizations were antagonized by VIP 10-28, but unaffected by alpha, beta-methylene ATP desensitization and Reactive Blue 2. 5. A single pulse of transmural nerve stimulation (2 ms, 15 mA) resulted in an inhibitory junction potential (IJP) that reached a maximal amplitude of 12.9 +/- 0.5 mV at 378 +/- 20 ms from the stimulus. This fast IJP was abolished by apamin (2 microM) or tetrodotoxin (1 microM), antagonized by alpha, beta-methylene ATP desensitization or Reactive Blue 2, but unaffected by VIP 10-28. 6. In the presence of apamin (1 microM), four pulses of transmural stimulation (2 ms, 20 Hz, 15 mA) resulted in an IJP that reached a maximal amplitude of 4.8 +/- 0.2 mV at 1.4 +/- 0.1 s from the stimulus. This slow IJP was antagonized by tetrodotoxin (1 microM) or VIP 10-28 (1 microM), augmented by Reactive Blue 2 (200 microM), and unaffected by alpha, beta-methylene ATP desensitization. 7. These findings provide evidence that both ATP and VIP are inhibitory neurotransmitters in the circular muscle layer of the ileum and that ATP may be the neurotransmitter responsible for the fast IJP and VIP the neurotransmitter responsible for the slow IJP.

Effects of vasoactive intestinal peptide on transmission from excitatory and inhibitory nerves to smooth muscle cells in chicken rectum

The effects of vasoactive intestinal peptide on electrical membrane properties and neuromuscular transmission, which is resistant to atropine and guanethidine, were investigated in isolated circular muscle strips from chicken rectum. Intracellular microelectrodes were used to record changes in membrane potential. Vasoactive intestinal peptide (0.2–1.0 μM) hyperpolarized the membrane of cells of the muscle strip. The mean maximal hyperpolarization produced by 1 μM vasoactive intestinal peptide was 5.3 ± 0.4 mV. Electronic potentials were unchanged in amplitude and the slopes of the current-voltage curves were similar before and after application of the peptide, indicating no change in membrane resistance in the presence of the peptide. The hyperpolarizing effect was preserved in the presence of apamin. These properties of the peptide-induced hyperpolarization were shared by the inhibitory junction potential. In addition, vasoactive intestinal peptide (0.5 or 1.0 μM) markedly inhibited or abolished the inhibitory junction potential but had little effect on the excitatory junction potential. The present results suggest a possible physiological implication of this peptide as a neurotransmitter of the inhibitory neurone in chicken rectum.

The multiple action of bradykinin on smooth muscle of guinea-pig taenia caeci

The action of bradykinin on the smooth muscle of guinea-pig taenia caeci was studied by measuring changes in membrane potential, the contractile state of the muscle cells and intracellular calcium concentrations at 22°C in the presence of tetrodotoxin, yohimbine, prazosin, propranolol and atropine. The bradykinin response was characterised by an initial hyperpolarization and suppression of spike activity followed by a sustained depolarization and an increased spike activity accompanied by inhibition of the phasic contractions, an increase in muscle tone and the development of phasic contractions, respectively. The actions of bradykinin were not affected after B 1-bradykinin receptors were blocked with des-Arg 9-[Leu 8]bradykinin. The events induced by bradykinin were accompanied by an increase in the intracellular calcium concentration, as monitored by quin-2 fluorescence. The hyperpolarization and depolarization persisted in the presence of diltiazem (10 −5 M) and in calcium-free conditions. The hyperpolarization could be evoked only once in the absence of calcium and was inhibited in the presence of apamin and after stimulation of α 1-adrenoceptors or P 2-purinoceptors. Membrane conductance was decreased during the sustained depolarization. The membrane depolarization was abolished after the sodium concentration gradient was reduced. These results show a multiple action of bradykinin mediated via B 2-receptors: (1) on calcium mobilization associated with activation of potassium channels; (2) on calcium release from intracellular stores and (3) on receptor-activated sodium channels.

The effect of noradrenaline on the ion permeability of isolated mammalian hepatocytes, studied by intracellular recording.

1. The influence of noradrenaline on the membrane potential and conductance of isolated guinea-pig and rabbit hepatocytes in short-term (2-8 h) tissue culture has been studied by intracellular recording. 2. Resting hepatocytes had linear current-voltage relationships, with input resistances of 166 and 216 M omega in guinea-pig and rabbit cells respectively. The recorded membrane potential was -18 mV in each species, though the true resting potential is likely to have been up to 10 mV greater. 3. The hepatocytes sometimes slowly hyperpolarized during intracellular recording, and this was associated with a fall in membrane resistance, and an increase followed by a decrease in membrane potential noise. These effects were abolished by quinine (200 microM) but not by apamin (50 nM), and are attributable to a K+ conductance activated by cell swelling. 4. Noradrenaline (2 microM, in the presence of propranolol at 1 microM) was applied to individual hepatocytes by pressure ejection (puffer pipette technique). After a short latency, the cells hyperpolarized by a mean of 18 mV in both guinea-pig and rabbit preparations. This was associated with a large rise in membrane conductance (50 nS in guinea-pig, 54 nS in rabbit cells). The reversal potential for this action was -38 mV. 5. The experiments were repeated in the presence of apamin (50 nM) to block the Ca2+-dependent K+ permeability which noradrenaline activates in these cells. Noradrenaline still caused some hyperpolarization and a substantial increase (approximately 40 nS) in conductance, with a reversal potential (Er) of -31 mV. This can be attributed to an increase in Cl- conductance. 6. In keeping with this interpretation, noradrenaline applied in the absence of Cl- (replaced by isethionate or gluconate) caused a much greater hyperpolarization (58 mV in guinea-pig, 40 mV in rabbit cells) associated with a smaller rise in conductance (approximately 12 nS). Er for this action was -95 mV (guinea-pig) and -68 mV rabbit), suggesting that the conductance increase was now mainly to K+. 7. The magnitudes of the conductance changes produced by noradrenaline under the various experimental conditions suggest that the increase in the conductance to Cl- (delta GCl) is 3-fold greater than that to K+ (delta GK). 8. The activation of delta GCl occurs either at the same time as delta GK, or (in ca. one cell in ten) a few seconds later.(ABSTRACT TRUNCATED AT 400 WORDS)

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

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.

Some Effects of Nicorandil on the Smooth Muscles of the Rat and Guinea Pig

In rat isolated portal vein, nicorandil (0.1-500 microM) abolished spontaneous tension waves and inhibited mechanical responses to norepinephrine (0.1-100 microM) and KCl (5-80 mM). Intracellular electrical recording showed that nicorandil (0.1-1 microM) abolished spontaneous multispike complexes and at higher concentrations (up to 500 microM) raised the membrane potential to approximately -90 mV. Using 86Rb as a K+-marker, nicorandil (5-500 microM) increased the 86Rb efflux rate coefficient. In rat isolated aorta, nicorandil (8-32 microM) inhibited mechanical responses to norepinephrine (0.125-100 microM) and KCl (5-80 mM), but had no effect on 86Rb exchange. In guinea pig isolated taenia caeci, nicorandil (4-64 microM) relaxed spontaneous mechanical tone and increased 86Rb efflux in the absence and presence of apamin, 100 nM. It is concluded that the inhibitory effects of nicorandil in portal vein and taenia caeci are mediated at least in part by a mechanism which involves the opening of apamin-insensitive, 86Rb-permeable K+ channels. In aorta, however, the opening of such channels was not detected, and the inhibitory effects of nicorandil in this tissue are associated with an, as yet, undefined mechanism.

Non-cholinergic excitatory and inhibitory junction potentials in the circular smooth muscle of the guinea-pig ileum.

Intracellular micro-electrodes have been used to record evoked non-cholinergic junction potentials from the muscle layers of strips of guinea-pig ileum cut parallel to the longitudinal muscle. Transmural stimulation with single pulses, 0.6 ms duration produced inhibitory junction potentials (i.j.p.s) in the circular muscle layer. In the circular muscle layer, transmural stimulation with repetitive volleys (e.g. three pulses, 0.6 ms, 50 Hz delivered every 4 s) at low stimulus strengths (25-40 mA) produced non-cholinergic excitatory junction potentials (e.j.p.s) after an initial i.j.p. At higher stimulus strengths (greater than 40 mA) i.j.p.s occurred following each volley but were superimposed on a prolonged depolarization. Following repetitive volley stimulation every four seconds in the presence of apamin (0.25 microM), the i.j.p.s. were abolished and the non-cholinergic e.j.p.s clearly showed facilitation. At higher stimulus strengths (and at volley repetition rates of less than 0.1 Hz) volley stimulation now produced an apamin-resistant slow hyperpolarization followed by a distinct slow depolarization. Electrotonic potentials were readily recorded from the superficially located longitudinal muscle cells: in this muscle layer transmural stimulation produced small, slow changes in membrane potential. These results suggest that non-cholinergic excitatory and inhibitory nerve fibres primarily supply the circular rather than the longitudinal muscle layer in the guinea-pig ileum.

The influence of α,β-methylene ATP on α1-receptor-operated channels in guinea-pig taenia caeci

The influence of the ATP analog α,β-methylene ATP on the action of adrenaline on α 1-receptors of smooth muscle cells of guinea-pig taenia caeci was studied by measuring potential changes. The preparation was superfused (1 ml/min) with Krebs solution or calcium-free solution containing atropine (10 −6 M) and propranolol (10 −6 M) at 22°C, using the sucrose-gap method. The ATP analog (10 −5 to 4 × 10 −4 M) and adrenaline (10 −5 M) both caused a transient hyperpolarization in the absence of external calcium (20 min). The response (area under the ‘curve’) evoked under calcium-free conditions (20 min) increased with the concentration of the ATP analog. The response was diminished when preceded by the adrenaline response (10 −5 M) or when evoked after repeated addition of the analog to the superfusate (35 min). The adrenaline response was also diminished when preceded by the ATP analog. The responses to the ATP analog or adrenaline in the presence of apamin (3 × 10 −7 M) in the absence of external calcium were characterized by depolarization of the muscle cells. Repeated addition of the ATP analog or adrenaline to the preparation under these conditions did not cause any effect. The results suggest strongly that adrenaline and α,β-methylene ATP both activate the same calcium-dependent process, producing calcium mobilization and the opening of apamin-sensitive potassium channels. Besides this action the ATP analog also activates apamin-sensitive potassium channels and this activation is independent of the availability of calcium in the adrenaline-sensitive pool.

Blockade of Ca-activated K conductance by apamin in rat sympathetic neurones.

Effects of apamin on rat sympathetic neurones were investigated by means of intracellular and extracellular recording. Apamin (50 nM) significantly shortened the after-hyperpolarization (AH) following the spike evoked by current injection and slightly decreased its peak amplitude without affecting the time course of the spike. The AH following the synaptically-evoked spike was also blocked by apamin. This effect was dose- and time-dependent (ID50 estimated by extracellular recording approximately 15 nM, 20 min after application) and poorly reversible. Transmission of a single volley was not affected by 50 nM apamin. Though a long depolarizing current caused one or two spikes in the cell, greater repetitive firing was observed in the presence of apamin. Spontaneous repetitive firing, however, was not observed except for anodal-break spikes. Resting potential and input membrane resistance were essentially unchanged by apamin. The maximum rate of rise of the Ca spike was not decreased by 50 nM apamin but the duration of the spike was lengthened by 60%. The AH following the Ca spike was also blocked by apamin. These results suggest that apamin suppressed the slow AH without any inhibition of the Ca flux into the cell and is useful as a blocker of GK(Ca) in the rat sympathetic neurone.

Antagonism of non-cholinergic excitatory junction potentials in the guinea-pig ileum by a substance P analogue antagonist

In the presence of atropine electrical transmural stimulation (using repetitive volleys, e.g. 3 pulses at 50 Hz applied every 4 s) of full thickness longitudinal strips of guinea-pig ileum produced non-cholinergic excitatory junction potentials (EJPs) and inhibitory junction potentials (IJPs) in the circular muscle layer. After abolition of the IJPs with apamin, the non-cholinergic EJPs clearly showed facilitation. In the presence of apamin and the substance P analogue antagonist, [ d-Arg 1, d-Pro 2, d-Trp 7,9,Leu 11]-substance P (SPA), the non-cholinergic EJPs were reduced by 60–90%; transmural stimulation now revealed an apamin-resistant IJP followed by a slow depolarization. The atropine-resistant EJPs are probably caused by the release of substance P (or a similar compound) and are likely to underlie the non-cholinergic contractions reported to occur in this tissue.

Pharmacological evidence for two types of Ca 2+-dependent K +-conductance in bullfrog sympathetic ganglion cells

The effect of apamin, a bee venom toxin, on the action potential and the spike afterhyperpolarization was studied in bullfrog sympathetic ganglion cells. Apamin reduced the duration of the afterhyperpolarization but did not affect the maximum rates of rise and fall of Na +- and Ca 2+-dependent action potentials. In the presence of apamin and Co 2+, the maximum rate of fall of the action potential was decreased, and the spike duration was prolonged. These results suggest that at least two types of Ca 2+-dependent K +-conductance co-exist in bullfrog sympathetic ganglion cells.

Effector mechanisms for α,β-methylene ATP and ATP derivatives in guinea-pig taenia caeci

The effects of the ATP analog α,β-methylene ATP and ATP derivatives on smooth muscle cells of guinea-pig taenia caeci were studied by measuring potential changes. The preparations were superfused (1 ml/min) with Krebs solution or calcium-free solution containing atropine (10 −6 M), prazosin (10 −6 M) and propanolol (10 −6 M) using the sucrose-gap method at 22°C. The ATP analog (10 −5 M to 4 × 10 −4 M) caused slowly developing hyperpolarization in the presence of calcium. Hyperpolarization also occurred on addition of the analog in calcium-free medium. The area of the response increased with the analog concentration (10 −5 M to 4 × 10 −4 M). The response evoked after repeated addition of the ATP analog had slightly lower amplitude and slower onset than the first response both in the presence and the absence of calcium. The area of the response to the ATP analog in the absence of calcium was also decreased when preceded by the ATP response. When the opposite experimental sequence was used the ATP response was strongly inhibited, when preceded by the analog response evoked by the same concentration. The ATP analog caused depolarization of the muscle cells in the presence of apamin (3 × 10 −7 M), which was also observed with ATP. The ATP derivatives (4 × 10 −4 M) caused hyperpolarization of the smooth muscle cell membrane, except hypoxanthine and inosine which were inactive. The responses evoked by the nucleotides were characterized by hyperpolarization reaching a maximum followed by a sustained component in calcium-free conditions, the latter being more pronounced with AMP than with ATP. The sustained hyperpolarization persisted during the second response or after a preceding ATP response. In the presence of apamin, initial depolarization was followed by sustained hyperpolarization. The response to adenosine was reflected by sustained hyperpolarization in both the absence and presence of calcium or apamin. The inhibitory junction potential elicited after electrical stimulation (2 s, 5 pps) of taenia caeci and the response evoked by exogeneously applied ATP, both reflected by hyperpolarization of the muscle cells, reached the same level in the presence or absence of α,β-methylene ATP. The results indicate that α,β-methylene ATP opens apamin-sensitive potassium channels in smooth muscle cells of taenia caeci directly and via a mechanism also activated by ATP and the ATP nucleotides ADP and AMP.

Responses evoked by electrical stimulation, adenosine triphosphate, adenosine and 4-aminopyridine in taenia caeci of the guinea-pig

Electrical stimulation of the guinea-pig taenia caeci (5 Hz, 2 s) caused enhancement of the [ 3H]purine flux from [ 3H]adenosine pools, accompanied by hyperpolarization (inhibitory junction potential) and relaxation of the muscle cells (35°C). Interaction with receptors sensitive to catecholamines and acetylcholine was prevented by phentolamine (10 −6 M), propranolol (10 −6 M) and atropine (10 −6 M), respectively. The hyperpolarization and relaxation were completely inhibited by tetrodotoxin (TTX; 3×10 −7 M) but a substantial part of the [ 3H]purine flux persisted. The flux from the [ 3H]purine pool, from the [ 3H]methylcholine pool and from the noradrenaline pool was enhanced in the presence of 4-aminopyridine (3×10 −4 M; 4-AP), known to facilitate transmitter release. The release from the [ 3H]methylcholine pool was limited by hemicholinium (HC-3; 5×10 −4 M) and the release of noradrenaline was limited by reserpine (5 mg/kg; 24 h). The excess release of [ 3H]purine caused by 4-AP was completely abolished in the presence of TTX in preparations treated with HC-3 and reserpine. Addition of 4-AP to the Krebs solution evoked contraction of the smooth muscle cells. This response was abolished in the presence of HC-3 or atropine. The relaxation was also observed in reserpinized preparations in the presence of HC-3 and was not inhibited by either phentolamine or propranolol but was abolished in the presence of TTX. Hyperpolarization and suppression of spike activity accompanied the relaxation induced by 4-AP in reserpinized preparations treated with HC-3. Comparable responses were evoked by electrical stimulation of taenia caeci, by adenosine triphosphate (4×10 −4 M; ATP) and by adenosine (4×10 −4 M) in these experimental conditions. These responses evoked by electrical stimulation and by ATP were reversed in the presence of apamin (3×10 −7 M); the effect was reflected by an increased spike activity and contraction of the muscle cells in contrast to the adenosine response. The 4-AP response and the inhibitory junction potential could not be evoked in the absence of extracellular calcium. These results provide evidence that purine release from nerve endings is facilitated by 4-AP. The effect is not necessarily associated with the release of acetylcholine or noradrenaline and the responses evoked by the purine or ATP in smooth muscle cells of taenia caeci show similar characteristics.

Interactions between receptors that increase cytosolic calcium and cyclic AMP in guinea-pig liver cells.

The action of agonists which increase the K+ permeability of liver cells was studied by using a K+-sensitive electrode to record the net movement of K+ between guinea-pig isolated hepatocytes and their suspension medium. Two types of agonist were examined. Type 1 comprised angiotensin II, ATP, noradrenaline and amidephrine, all of which are thought to raise cytosolic Ca2+ in hepatocytes. The Type 2 agonists were isoprenaline and glucagon, which activate adenylate cyclase. Each type of agonist initiated K+ loss from the hepatocytes though the response to Type 2 agonists was more variable than that to Type 1, and sometimes absent. Simultaneous application of a small concentration of an agonist from each class caused a loss of K+ which was much larger than the sum of that seen with each agonist alone, i.e. potentiation occurred. The alpha-adrenoceptor antagonist, WB 4101, abolished potentiation if applied after an alpha-agonist, and before a Type 2 agonist, showing that both receptors have to be active for potentiation to occur. Simultaneous application of a maximal concentration of each type of agonist caused a larger loss of K+ (approximately 17% of the cell total within 45 s) than did a maximal concentration of a Type 1 agonist alone (approximately 10%). Since the K+ loss caused by these agonists is thought to be a consequence of a rise in cytosolic Ca2+, the influence of both types of agonist on 45Ca and 42K efflux from guinea-pig liver slices was studied. The effect of isoprenaline on 45Ca and 42K efflux became much greater following a previous application of the alpha-adrenoceptor agonist, amidephrine. In the presence of apamin, the potentiated effect of isoprenaline on 42K efflux was greatly reduced whereas that on 45Ca efflux was little affected. The effects of Type 1 and Type 2 agonists separately and together on the cyclic AMP content of isolated hepatocytes were examined. Type 2 agonists increased cyclic AMP in the expected way. The increase became slightly smaller, if anything, when a Type 1 agonist was applied at the same time. Hence potentiation could not be ascribed to changes in cyclic AMP formation. Possible mechanisms for potentiation are discussed. Our evidence suggests, albeit indirectly, that it is a consequence of an interaction between the effects of the two types of agonist on cytosolic Ca2+.

Apamin-resistant post-stimulus hyperpolarization in the circular muscle of the guinea-pig ileum

Transmural nerve stimulation of the guinea-pig ileum in the presence of atropine at 30°C has been found to produce a relatively brief (duration ∼ 1 s) inhibitory junction potential (IJP) followed by a longer lasting depolarization [1]. When short volleys of stimuli (e.g. 3 pulses, 0.5 ms, 10 Hz) were applied to this tissue, it was noted that the post-stimulus depolarization consisted of at least two distinct phases [2]. The first of these phases, which we called fast post-stimulus depolarization, peaked about 1–1.5 s following the stimulus, whereas the second phase (slow post-stimulus depolarization) peaked about 2–3 s later [2]. After prolonged exposure to substance P (5 × 10 −7 M for > 15 min) the first depolarizing phase was abolished, whereas the second depolarizing phase appeared to be enhanced [4]. In the earlier series of experiments [1], using single stimuli, we observed that the bee-venom neurotoxin apamin [3] abolished the IJP and only a single depolarizing junction potential remained. The time-to-peak of this non-cholinergic excitatory junction potential correlated with the time-to-peak of the initial fast depolarizing response observed when volleys of stimuli were used. In the present series of experiments we have re-examined the effects of apamin on the membrane potential responses following short volleys of stimuli and also the effects of prolonged exposure to substance P in the presence of apamin. We report here, that, following abolition of the IJP by exposure to apamin, prolonged exposure to substance P abolished the fast post-stimulus depolarization and revealed a slow hyperpolarizing response.

The effects of vasoactive intestinal polypeptide on the electrical activity of guinea-pig intestinal smooth muscle

The effect of vasoactive intestinal polypeptide (VIP) on the electrical activity of the smooth muscle of the guinea-pig taenia coli has been examined using the single sucrose gap apparatus. VIP usually caused a slowly developing, long-lasting membrane hyperpolarisation, although sometimes it reduced the frequency of spontaneous spike discharge without hyperpolarising the membrane. In contrast, non-adrenergic, non-cholinergic nerve stimulation initiated rapid membrane hyperpolarisation followed by post inhibitory excitation. In the presence of apamin, the VIP response was little affected, whereas the responses to non-adrenergic, non-cholinergic nerve stimulation were either markedly antagonised or reversed to membrane depolarisation. Consideration is given to the possible role of either VIP or ATP as the neurotransmitter responsible for the inhibitory junction potential elicited in the guinea-pig taenia coli in response to non-adrenergic inhibitory nerve stimulation.

The nature of non-cholinergic, non-adrenergic transmission in longitudinal and circular muscles of the guinea-pig ileum.

1. The nature of the non-cholinergic, non-adrenergic (non-ch., non-adr.) excitatory and inhibitory transmission in the longitudinal and circular muscle layers of the guinea-pig ileum was investigated, and the effects of various agents on the junction potentials were observed using the micro-electrode method.2. In longitudinal muscle cells, ATP (3 x 10(-5)-10(-4) M) and adenosine (10(-5)-10(-4) M) depolarized the membrane, decreased the input resistance, increased the spike activity and abolished the generation of cholinergic excitatory junction potentials (e.j.p.s).3. In the presence of atropine (10(-6) M) with guanethidine (10(-5) M), field stimulation evoked three different types of the response (non-ch., non-adr. e.j.p.s, i.j.p.s (inhibitory junction potentials) or both) from cells of the longitudinal muscle layers, and only one type of the response (non-ch., non-adr. i.j.p.s) from cells of the circular muscle layer. In the following experiments atropine and guanethidine were present in the bathing fluid for at least 20 min.4. In some longitudinal muscle cells (non-ch., non-adr. i.j.p. type), ATP (5 x 10(-6)-10(-3) M) and adenosine (10(-5)-3 x 10(-5) M) depolarized the membrane, while in other cells (non-ch., non-adr. e.j.p. type), ATP (10(-5)-10(-4) M) and adenosine (10(-5)-3 x 10(-5) M) hyperpolarized the membrane and further increases in the concentration of ATP (10(-3) M) resulted in a depolarization of the membrane.5. Apamin (10(-7)-3 x 10(-6) M) inhibited the generation of non-ch., non-adr. i.j.p.s in both longitudinal and circular muscle cells, while this agent had no effect on the non-ch., non-adr. e.j.p.s. As a consequence, in some cells of the longitudinal muscle layer (non-ch., non-adr. e.j.p. and i.j.p. type) the amplitude of e.j.p.s was enhanced in the presence of apamin. TEA (5 x 10(-3)-1.5 x 10(-2) M) suppressed the after-hyperpolarization of the spike and i.j.p.s recorded from both muscle layers, whereas the duration and amplitudes of cholinergic and non-ch., non-adr. e.j.p.s were enhanced.6. Vasoactive intestinal polypeptide (VIP; 10(-8)-10(-7) M) had no effect on the membrane potential and junction potentials of longitudinal and circular muscle layers.7. Substance P (SP; 10(-8)-10(-7) M) depolarized the membrane of cells of the longitudinal layer (non-ch., non-adr. e.j.p. type), while this agent had no effect on cells of longitudinal (non-ch., non-adr. i.j.p. type) and circular muscle layers. SP suppressed the generation of non-ch., non-adr. e.j.p.s but had no effect on i.j.p.s. Generation of non-ch., non-adr. e.j.p.s was not restored under conditions of repolarization of the membrane to the resting level by application of inward current.8. Bradykinin (BK; 10(-8)-10(-5) M) hyperpolarized the membrane and suppressed the generation of i.j.p.s in the cells of longitudinal (non-ch., non-adr. i.j.p. type) and circular muscle layers. However, when the membrane potential was displaced to the control level by outward current in the presence of BK, field stimulation evoked the i.j.p. In cells of non-ch., non-adr. e.j.p. type of the longitudinal muscle layer, BK depolarized the membrane, increased the spike activity, generated slow waves and blocked the generation of non-ch., non-adr. e.j.p.s. Displacement of the membrane potential to the control level by inward current did not restore the non-ch., non-adr. e.j.p.s.9. These results suggest that in the guinea-pig ileum ATP and adenosine probably do not contribute to the generation of non-ch., non-adr. e.j.p.s and i.j.p.s, as transmitter substances. The actions of other possible candidates such as SP and BK, are discussed.

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. Apamine 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 apamine. Reduction of the extracellular chloride or sodium concentration, blockade of the nervous system by TTX (3 × 10 −7 M) or inhibition of spike activity by D600 (3 × 10 −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.