The bradykinin potentiating activity of two pentapeptides on various isolated smooth muscle preparations.

Two bradykinin potentiating peptides A-VI5 (Val-Glu-Ser-Ser-Lys) and BPP5a (Pyr-Lys-Trp-Ala-Pro), were compared with respect to their potentiation of a number of different bradykinin effects on six isolated smooth muscle preparations. Apart from the considerable difference in effective concentrations, no essential qualitative difference was observed between these two peptides. Therefore the assumption of a different mechanism of action for the two peptides, which have a completely different structure, could not be substantiated.     

Effects of neuronal polypeptides on intestinal smooth muscle; a comparison with non-adrenergic, non-cholinergic nerve stimulation and ATP.

Substance P, somatostatin, enkephalin and vasoactive intestinal polypeptide (VIP) did not mimic the inhibitory responses to non-adrenergic, non-cholinergic nerve stimulation. Substance P (0.1-10 microgram/ml) always caused contraction, enkephalin (0.1-10 microgram/ml) and somatostatin (0.1 microgram/ml) were inactive, while VIP (0.1-1 microgram/ml) produced very slow relaxation, taking about 4 min to reach a maximum after a latency of about 60 sec. Low concentrations of neurotensin (1-10 ng/mg) caused contraction, but at higher concentrations (50-1000 ng/ml) it produced a biphasic response which consisted of an initial contraction followed by a slow relaxation. In high tone preparations, the slow relaxation did not mimic the nerve-mediated response, taking approximately 43 sec. to reach maximum, after a long latency of about 15 sec. In contrast, ATP (0.1-50 microgram/ml) mimicked closely the rapid responses to non-adrenergic, non-cholinergic nerve stimulation in all preparations, whether the tone was low, medium or high. The time for the inhibitory response to reach maximum was about 15 sec after a latency of approximately 1 sec. Indomethacin (3.4-34 microgram/ml) did not unmask any inhibitory responses to any of the peptides. It is concluded that ATP remains the most likely substance to be the inhibitory transmitter released from non-adrenergic, non-cholinergic nerves supplying the smooth muscle of the taenia coli.     

Direct evidence for ATP release from non-adrenergic, non-cholinergic ("purinergic") nerves in the guinea-pig taenia coli and bladder

Demonstration of release of ATP from smooth muscle preparations during stimulation of purinergic nerves is complicated by the difficulty in showing whether it comes from nerve or muscle. ATP released during relaxation of the guinea-pig taenia coli and contraction of bladder strips in response to purinergic nerve stimulation was measured in the superfusate using the luciferin-luciferase ATP assay method. The amount of ATP increased 2-6 fold during isometric responses to purinergic nerve stimulation. This release was blocked by tetrodotoxin but not by adrenergic nerve destruction with 6-hydroxydopamine. No significant release of ATP was detected during comparable responses elicited by direct muscle stimulation. These results provide further support for the purinergic nerve hypothesis.     

The mechanism of action of two bradykinin-potentiating peptides on isolated smooth muscle.

Bradykinin-induced contractions in the guinea-pig ileum were potentiated by the peptides A-VI-5 (Val-Glu-Ser-Ser-Lys) and BPP5a (Pyr-Lys-Trp-Ala-Pro), while the contractions induced by other agonists were not affected. Neither peptide added alone caused any response. Previous addition of the peptides shortened the latent period following the addition of bradykinin to a value corresponding to the contraction height with an equivalent dose of bradykinin added alone. Bradykinin in contact with a piece of ileum was inactivated at a relatively slow rate. This inactivation was not inhibited by either A-VI-5 or BPP5a in doses causing potentiation. Suppression of the cholinergic activity by cooling, atropine, morphine or tetrodotoxin did not influence the potentiating activity. Addition of the peptides at the moment a submaximal contraction due to bradykinin had been fully established, increased the contraction height within seconds. The two peptides caused a parallel shift to the left of the dose-effect curve of bradykinin, whereas the maximum bradykinin effect remained unchanged. It is concluded that sensitization of bradykinin receptors due to an increased affinity of the receptor for bradykinin is the hypothesis which best fits the experimental findings.     

Apamin,a nonspecific antagonist of smooth muscle relaxants

Summary Apamin, a peptide of bee venom, was shown to inhibit the relaxant responses of guinea-pig taenia caeci to ATP, noradrenaline, adenosine and, less effectively, to stimulation of noradrenergic inhibitory nerves. Thus apamin acts nonspecifically and, contrary to the suggestion of Vladimirova and Shuba (1978), the fact that inhibitory responses due to nerve stimulation and ATP are blocked by the toxin does not allow conclusions as to the possible transmitter role of ATP in these nerves.     

Effects of the immunosuppressant cyclosporin A on neurotransmitter release from peripheral non-adrenergic, non-cholinergic nerves

This study was undertaken to determine the effect of the immunosuppressant cyclosporin A on neurotransmitter release from non-adrenergic, non-cholinergic nerves (tachykininergic nerves) in the rabbit iris sphincter muscle. Cumulative application of cyclosporin A (0.1 to 10 μM) caused a slow onset of contraction in a concentration-dependent manner. Both FK888 (1 μM) and capsaicin (10 μM), a substance P receptor antagonist and a substance P-depleting agent, respectively, inhibited the contractile effect of cyclosporin A, whereas atropine (1 μM) had no effect. Both cyclosporin A and capsaicin (10 μM) stimulated the release of substance P-like immunoreactivity in the iris. Neither the sodium channel blocker tetrodotoxin (1 μM), the N-type voltage-dependent Ca²⁺ channel blocker ω-conotoxin GVIA (1 μM) nor the P-type channel blocker ω-agatoxin IVA (0.2 μM) affected cyclosporin A (1 μM)-induced contraction. In contrast, the L-type Ca²⁺ channel blocker nicardipine (10 μM) inhibited this contractile effect. These results suggest that cyclosporin A stimulates substance P-like tachykinin release by activating L-type voltage-dependent Ca²⁺ channels, resulting in contraction of the rabbit iris sphincter muscle. Received: 4 March 1997 / Accepted: 4 June 1997     

Evidence that prostaglandin is responsible for the 'rebound contraction' following stimulation of non-adrenergic, non-cholinergic ('purinergic') inhibitory nerves.

The prostaglandin synthesis inhibitor, indomethacin, blocks the "rebound contractions" which characteristically follow the inhibitory responses of the guinea-pig taenia coli to non-adrenergic, non-cholinergic (purinergic) nerves and to exogenously applied ATP, without affecting the responses to periaterial adrenergic nerves. Since adenine nucleotides are known to induce prostaglandin synthesis, this result is consistent with the purinergic hypothesis and suggests that purinergic nerves may form a link with prostaglandin in the physiological regulation of a variety of organs.     

The effect of enkephalins on the intramural inhibitory non-adrenergic nerve responses of smooth muscle.

The study concerned the effect of enkephalins and naloxone on the inhibitory non-adrenergic nerve responses of the taenia coli and the stomach smooth muscle cells of the guinea pig. Met-enkephalin, leu-enkephalin and D-ala-enkephalin (10(-8)10(-5)M) did not affect the muscle tension or membrane potential measured simultaneously using the sucrose-gap method. Naloxone (5x10(-8)--5x10(-6)M) relaxed the stomach strip, but did not modify the inhibitory junction potential. These results indicate that enkephalins are not involved in the nonadrenergic inhibitory nervous system, except possibly as neuromodulators at an interneuronal synaptic level.     

The effect of catecholamines and ATP on the smooth muscle cell membrane of the guinea-pig taenia coli

A comparative study was made of the effects of some catecholamines and purine-like compounds on the smooth muscle cell membrane of the guinea-pig taenia coli, using the double sucrose-gap method. The inhibitory junction potential (IJP) might be mediated by purinergic or adrenergic nerves. Therefore, the effects of these compounds on the membrane resistance and the membrane potential and also on the IJP were measured in the presence of guanethidine and atropine. The potency of the catecholamines tested to mimic the IJP with regard to membrane hyperpolarization and decrease of membrane resistance suggested an action on α-adrenoceptors: adrenaline > noradrenaline ? isoprenaline. Equipotent concentrations of adrenaline and adenosine triphosphate (ATP) were found to exert the same hyperpolarization of the smooth muscle membrane in both the presence and absence of chloride. From the effects observed with phentolamine and tolazoline it is concluded that the IJP is not mediated by adrenergic nerves, for the adrenaline response was abolished, whereas the IJP was unaffected. Furthermore, the ATP-blocking potencies of phentolamine and imidazole could not be demonstrated. On the other hand, the palallelism observed between the responses obtained with ATP and with transmural stimulation is in favor of the hypothesis that the IJP is mediated by purinergic nerves.     

Multiple inhibitory mechanisms mediate non-adrenergic non-cholinergic relaxation in the circular muscle of the guinea-pig colon

Summary The mechanisms responsible for nerve-mediated, non-adrenergic, non-cholinergic (NANC) relaxation in mucosa-free circular muscle strips from the proximal colon of the guinea-pig were investigated. Electrical field stimulation (EFS, 1–20 Hz, trains of 5 s duration, 100 V, 0.25 ms pulse width) in the presence of atropine (1 mol/l) and guanethidine (3 mol/l) evoked a triphasic motor response consisting of. (a) a primary relaxation, (b) a rebound contraction and (c) a secondary relaxation. These three responses were abolished by tetrodotoxin (1 mol/l). Both apamin (0.01–0.3 mol/l), a known blocker of low conductance, calcium-activated potassium channels in smooth muscles, and L-nitroarginine (L-NOARG) (1–100 mol/l), a known blocker of nitric oxide (NO) synthase, increased the tone of the strips. Maximum effects on tone were observed with 0.1 mol/l apamin (21 ± 3% of KCl-induced contraction) and 30 mol/l L-NOARG (26 ± 4% of KCl response). The combined administration of 0.1 mol/l apamin and 30 mol/l L-NOARG produced an increase in tone (47 ± 5% of KCl response) that was larger than that produced by either compound alone. Neither apamin (0.1 mol/l) nor L-NOARG (30 mol/l) affected the isoprenaline-induced relaxation.Apamin (0.1 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies without affecting the secondary relaxation. Apamin also enhanced the rebound contraction at a frequency of 1 Hz. L-NOARG (30 mol/l) depressed, but did not abolish, the primary relaxation to EFS at all frequencies, had no effect on the rebound contraction and inhibited the secondary relaxation evoked at frequencies of 1–5 Hz, but not 10–20 Hz. L-arginine (300 mol/l) reversed the effect of L-NOARG on tone and the inhibitory effect on the EFS-evoked relaxation. In the presence of apamin and L-NOARG, the primary relaxation was suppressed at all frequencies; the secondary relaxation was inhibited at 1–5 Hz and unchanged at 10–20 Hz, as observed with L-NOARG alone. We conclude that three distinct mechanisms mediate the NANC relaxation of the circular muscle of the proximal colon of the guinea-pig in response to EFS. One mechanism can be operationally defined as apamin-sensitive and a second as L-NOARG-sensitive, the latter implying a possible role of NO as an inhibitory transmitter. A third NANC inhibitory mechanism, which is apamin- and L-NOARG-resistant, is also suggested.Correspondence to: C. A. Maggi at the above address     

Effect of temperature on the transmitter release from the "purinergic" nerves in the guinea-pig taenia coli

Effects of temperature on the inhibitory junction potential (IJP) mediated via intramural ‘purinergic’ nerves were studied. The IJP area and the membrane conductance of the smooth muscle cell hardly changed between 16 and 28°C. The amplitude, stimulus-top time and decay time of the IJP however, changed linearly with temperature (with +0.5 mV, ?0.2 sec and ?0.3 sec per °C respectively). A temperature dependent synchronization of transmitter release can account for the observed phenomena.     

Effect of stretching on the sensitivity of the guinea pig ileum to bradykinin and on its modification by bradykinin potentiating peptides

The sensitivity of the guinea pig isolated ileum to bradykinin, but not to other agonists, was increased ca. 2-fold during the 3–4 h following mounting of the preparation under 1 g load. Concomitantly, a decrease was observed in the bradykinin potentiating effect of BPP9a, and of potentiator B but not potentiator C. This decrease was observed only with analogues of BPP9a or potentiator B which retained one or both of the basic amino acid residues in these peptides. Similar stretching of the rat isolated uretus did not affect bradykinin sensitivity or the potency of bradykinin potentiating peptides. Kininase activity of the ileum significantly increased during the 4 h period after mounting of the loaded preparation, but was not affected by treatment with BPP9a. It is proposed that bradykinin sensitivity is favoured by the changes in sodium and potassium transport in the cell membrane caused by stretching of the ileum, and that a similar mechanism may be partly responsible for the action of bradykinin potentiating peptides.     

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.     

Involvement of both L- and N-type voltage-dependent Ca2+ channels in KCl- and veratridine-evoked transmitter release from non-adrenergic, non-cholinergic nerves in the rabbit iris sphincter muscle

To determine which types of voltage-dependent Ca²⁺ channels mediate tachykinin release in the isolated rabbit iris sphincter muscle, we examined the effects of several Ca²⁺ channel modulators on contractions induced by either an elevation of the extracellular KCl concentration or application of the Na⁺ channel activator veratridine. Contractions caused by either 45.9mMKCl or veratridine (10μM) were inhibited by spantide (10μM), a tachykinin receptor antagonist, and capsaicin (10μM), a tachykinin-depleting agent, but were not changed by atropine. Nicardipine, an L-type Ca²⁺ channel blocker, inhibited contractions induced by KCl and veratridine in a concentration-dependent manner. ω-Conotoxin GVIA (1μM), an N-type Ca²⁺ channel blocker, inhibited only contractions induced by lower concentrations of KCl, both when applied alone and when combined with nicardipine. Bay K8644, an L-type Ca²⁺ channel activator, caused a spantide- and nicardipine-sensitive contraction in muscles partially depolarized with 15.9mMKCl, and enhanced contractions induced by 15.9mMKCl and veratridine (2μM). ω-Agatoxin IVA (0.3μM), a P-type voltage-dependent Ca²⁺ channel blocker, did not affect contractions induced by either KCl or veratridine. Contractions induced by exogenous substance P were not modified by any of the Ca²⁺ channel blockers or by Bay K8644. These results suggest that, in the rabbit iris sphincter muscle, L- and N-type voltage-dependent Ca²⁺ channels are involved in neurotransmitter release from tachykininergic nerves elicited by high KCl and by veratridine. Received: 21 May 1996 / Accepted: 14 January 1997     

ATP release caused by bradykinin, substance P and histamine from intact and cultured smooth muscles of guinea-pig vas deferens

Histamine (60 μM) produced ATP release from segments of guinea-pig vas deferens which was blocked by pyrilamine and triprolidine, H₁-blockers, but not by ranitidine, an H₂-blocker. The evoked-release was inhibited by the mitochondrial inhibitors, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and oligomycin. Bradykinin (BK) and substance P (SP) also caused substantial and moderate release of ATP, respectively. The BK-evoked release of ATP was inhibited by HOE140, a B₂-antagonist, but not by [Des-Arg¹⁰] HOE140, a B₁-antagonist. On the other hand, VIP, angiotensin II (AII) and cholecystokinin-octapeptide (CCK-8) failed to elicit a measurable release of ATP. Histamine and BK also enhanced the release of ATP from superfused cultured smooth muscle cells. These results suggest that ATP may be released as an autacoid from the smooth muscles in the presence of these chemical mediators. Received: 13 August 1997 / Accepted: 26 November 1997     

The different mechanisms of action of nicorandil and adenosine triphosphate on potassium channels of circular smooth muscle of the guinea-pig small intestine

Summary Nicorandil (10 mol/l–0.3 mmol/l) and ATP (1 mol/l–0.1 mmol/l) hyperpolarized the membrane of circular smooth muscle of the guinea-pig small intestine and increased conductance of the membrane probably to K ions as estimated by the effect on the current-voltage relationship. In the presence of a maximally hyperpolarizing concentration of nicorandil (0.1 mmol/l), ATP produced a further hyperpolarization of 5 mV. The ATP-induced but not the nicorandil-induced hyperpolarization required the presence of Ca in the medium, and the ATP-induced hyperpolarization was blocked by apamin treatment (1 nmol/l) or by MnCl₂ (1.3 mmol/l). On the other hand, both hyperpolarization responses were blocked by the local anaesthetics procaine (0.1–1 mmol/l), lidocaine (0.1–1 mmol/l) or cocaine (0.3–1 mmol/l), with different potencies. Field stimulation of smooth muscle of the small intestine produced inhibitory junction potentials (i.j.p.s) and these were inhibited by apamin (10 nmol/l–100 nmol/l). In the presence of ATP, the amplitude of the i.j.p.s was markedly reduced, but in the presence of nicorandil the amplitude was only slightly reduced, consistent with the same increase in ionic conductance and hyperpolarization of the membrane. These results indicate that ATP and nicorandil hyperpolarize the membrane by activating different K-channels, i.e. Ca dependent and Ca insensitive K channels, respectively. As assessed from the effects of local anaesthetics and the membrane properties, the circular muscle may also possess other K channels different from the ATP and nicorandil sensitive K channels.     

A comparison of the effects of angiotensin II and heptapeptide on smooth muscle (vascular and uterine).

On the rabbit isolated aorta, dose-dependent contractions to both angiotensin II and heptapeptide ([des-Asp1]-angiotensin II) were obtained. The curves were parallel, and reached the same maximum level. On the rat isolated uterus, angiotensin II and the heptapeptide displayed non-parallel dose-response curves. Results obtained with angiotensin-analog antagonists and cross-tachyphylaxis experiments suggest that the heptapeptide and angiotensin II act, preferentially, on different populations of receptors in the uterus. The difference in action of indomethacin on recovery from tachyphylaxis to angiotensin II and heptapeptide on rat isolated aorta suggests that the mechanism of induction of tachyphylaxis by these two peptides may differ. SQ 20881, the angiotensin converting enzyme inhibitor, totally inhibited uterine responses to both decapeptide and nonapeptide, while slightly potentiating those to angiotensin II and heptapaptide. Indomethacin had no significant effect on uterine responses to either angiotensin II or the heptapeptide.     

Antagonism of the effects of purinergic nerve stimulation and exogenously applied ATP on the guinea-pig taenia coli by 2-substituted imidazolines and related compounds

2-Substituted imidazolines and structurally related compounds, have been tested as possible antagonists to the inhibitory actions of ATP and purinergic nerve stimulation in the guinea-pig taenia coli.Imidazole at neutral pH had no effect on these responses. High concentrations of phentolamine, antazoline, tolazoline and yohimbine strongly reduced the inhibitory responses to ATP and purinergic nerve stimulation but not the inhibitory responses to amyl nitrite. These results are consistent with the purinergic nerve hypothesis. However, it is unlikely that the imidazoline drugs are causing specific blockade of purinergic transmission since they are known to have a wide range of pharmacological actions.     

The contribution of calcium and potassium to the alpha-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.     

A comparison of the excitatory and inhibitory effects of non-adrenergic, non-cholinergic nerve stimulation and exogenously applied ATP on a variety of smooth muscle preparations from different vertebrate species

1. The responses to non-adrenergic, non-cholinergic nerve stimulation have been compared with those to exogenously applied ATP on seventeen different tissues from a number of vertebrate classes.2. Stimulation of all the mammalian gut preparations studied (with the exception of the guinea-pig ileum) after blockade of the effects of adrenergic and cholinergic nerve stimulation by guanethidine (3.5 muM) and hyoscine (1.3 muM) caused inhibition; exogenously applied ATP mimicked this inhibitory response.3. Stimulation of the guinea-pig ileum in the presence of hyoscine and guanethidine, usually caused a diphasic response, relaxation followed by contraction; exogenously applied ATP mimicked this response, in contrast to acetylcholine and noradrenaline which caused excitation and relaxation respectively.4. Stimulation of preparations of lower vertebrate gut and guinea-pig bladder in the presence of hyoscine and guanethidine caused contraction; exogenously applied ATP mimicked this contractile response.5. In each preparation the time course of the response to ATP was similar or identical to the response to non-adrenergic, non-cholinergic nerve stimulation.6. The results are consistent with the hypothesis that a purine nucleotide may be the transmitter substance released from non-adrenergic, non-cholinergic nerves supplying smooth muscle preparations from a number of vertebrate classes.