A small-conductance charybdotoxin-sensitive,apamin-resistant Ca2+-activated K+ channel in aortic smooth muscle cells (A7r5 line and primary culture)

A small conductance K⁺ channel was identified in smooth muscle cells of the rat aortic cell line A7r5 and also in rat aortic smooth muscle cells in primary culture, using conventional single-channel recording techniques. The single-channel conductance shows no rectification, either in the range –70 to +40 mV under asymmetrical conditions (9.1 pS), or in the range –100 to +50 mV in symmetrical 150 mM K⁺ (37 pS). Channel activity is reversibly inhibited by extracellular application of charybdotoxin, with a concentration of 8 nM producing half-maximal inhibition. It is unaffected by apamin or scyllatoxin. Channel activity depends on the presence of free Ca²⁺ on the cytosolic face of the membrane, with an activation zone between 0.1 and 1 M. This small-conductance, charybdotoxin-sensitive, Ca²⁺-regulated K⁺ channel is activated by vasoconstrictors such as vasopressin and endothelin.     

Apamin distinguishes two types of relaxation mediated by enteric nerves in the guinea-pig gastrointestinal tract

Summary Eight smooth muscle preparations from the stomach, small intestine and large intestine of the guinea-pig were used to compare apamin's actions in reducing the effectiveness of transmission from enteric inhibitory nerves and in reducing responses to inhibitory agonists ,-methylene ATP, VIP and isoprenaline. The effects of apamin on inhibitory reflexes in the ileum and colon were also evaluated. Apamin had little or no effect on responses to VIP and isoprenaline in any region, but consistently and substantially reduced responses to ,-methylene ATP. Responses to stimulation of enteric inhibitory neurons were substantially reduced by apamin in the antrum circular muscle, ileum longitudinal and circular muscle, taenia coli and distal colon longitudinal muscle, but it was ineffective in the fundus circular muscle, proximal colon longitudinal muscle and distal colon circular muscle. It caused a small reduction of the relaxation of the ileal circular muscle caused reflexly by distension, but did not modify the similar descending inhibitory reflex in the circular muscle of the colon. It is concluded that apamin can be used to distinguish two types of non-noradrenergic transmission from enteric inhibitory nerves to gastrointestinal muscle. Furthermore, neither VIP nor ATP can be the sole transmitter chemical released from enteric inhibitory neurons throughout the gastrointestinal tract.     

A comparison between ATP and bradykinin as possible mediators of the responses of smooth muscle to non-adrenergic non-cholinergic nerves

The effects of ATP, bradykinin (BK) and electrical stimulation of intramural non-adrenergic non-cholinergic nerves (NS) were compared in four smooth muscle preparations. In the guinea-pig taenia caeci and rat duodenum, ATP (10^?7-5 × 10^?5 M) and BK (5 × 10^?10-10^?7 M) closely mimicked the response to NS. The relaxations to BK, but not to ATP or NS, were inhibited by carboxypeptidase B (3–15 U/ml) and apamin (10^?8-5 × 10^?8 M) prevented the relaxations to all three stimuli. BK contracted the guinea-pig distal colon whereas ATP and NS caused inhibition. In the guinea-pig bladder, ATP and NS induced rapid phasic contractions whereas BK caused tonic contractions. In the latter two preparations, incubation with indomethacin failed to reveal any BK relaxationic In view of its failure to mimic the nerve-mediated response in two of the tissues, and of its selective inhibition by carboxypeptidase B in the other two, BK is less likely than ATP to be the transmitter in non-adrenergic non-cholinergic nerves supplying smooth muscle.     

Participation of nitric oxide in the relaxation of the rat gastric corpus

Nitric oxide is an important mediator of the relaxation in the rat gastric fundus. The present study investigates the role of NO in the rat gastric corpus in vitro, since the corpus differs from the fundus with regard to its physiological function and its spontaneous motor behaviour.In the presence of guanethidine electrically induced relaxations of circular, mucosa-free corpus strips precontracted with bethanechol were concentration-dependently reduced by the NO-synthase inhibitors l-N^G-nitro-arginine (l-NNA) or l-N^G-nitro-argininemethyl-ester (l-NAME). The d-enantiomers were markedly less active. The inhibitory effect of l-NAME could be prevented by l-arginine. l-NNA and l-NAME, however, did not influence spontaneous motility or the bethanechol-induced contraction. Vasoactive intestinal polypeptide or sodium nitroprusside also relaxed the muscle strips, but these relaxations were not affected by l-NAME. When the corpus strips were stimulated electrically from baseline, they reacted with a contraction followed by relaxation. l-NNA or l-NAME blocked the relaxatory and enhanced the contractile component. In strips that also reacted with a rebound contraction, it was blunted by l-NAME. These effects of the NO-synthase inhibitors were abolished in the presence of atropine. Apamin increased the electrically induced contraction of the muscle strips. Inhibition of the relaxation together with a further shift to contraction could only be seen when apamin was combined with l-NNA. The inhibitory action of apamin and apamin + l-NNA was not influenced by atropine.The results demonstrate a role of NO in the relaxation of the circular muscle of the rat gastric corpus both at a postsynaptic site and via inhibition of acetylcholine release. The relaxation induced by vasoactive intestinal polypeptide does not involve NO.     

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.     

Chemical characterization,antioxidant, anti-inflammatory and cytotoxic properties of bee venom collected in Northeast Portugal

Bee venom (BV) or apitoxin is a complex mixture of substances with reported biological activity. In the present work, five bee venom samples obtained from Apis mellifera iberiensis from the Northeast Portugal (two different apiaries) were chemically characterized and evaluated for their antioxidant, anti-inflammatory and cytotoxic properties. The LC/DAD/ESI-MSⁿ analysis of the samples showed that melittin was the most abundant compound, followed by phospholipase A2 and apamin. All the samples revealed antioxidant and anti-inflammatory activity but without a direct relation with any of the individual chemical components identified. The results highlight that there are specific concentrations (present in BV5) in which these compounds are more active. The BV samples showed similar cytotoxicity for all the tested tumour cell lines (MCF-7, NCI-H460, HeLa and HepG2), being MCF-7 and HeLa the most susceptible ones. Nevertheless, the studied samples seem to be suitable to treat breast, hepatocellular and cervical carcinoma because at the active concentrations, the samples were not toxic for non-tumour cells (PLP2). Regarding the non-small cell lung carcinoma, BV should be used under the toxic concentration for non-tumour cells. Overall, the present study corroborates the enormous bioactive potential of BV being the first report on samples from Portugal.     

Differential effects of potassium channel blockers on the activity of the locomotor network in neonatal rat

The role played by various K+ channels during locomotor activity was studied using an in vitro neonatal rat spinal cord preparation. Locomotor-like activity was elicited by bath-applying serotonin (5-HT) and N-methyl-d-l-aspartate (NMA). Four different K+ channel blockers were tested by adding them to the superfusing saline. Each of the K+ channel blockers elicited a characteristic motor pattern with specific temporal parameters. Cs+ and tetraethyl ammonium both decreased the motor period, but had opposite effects on the burst amplitude. Apamin increased both the motor period and the burst amplitude. A dose-response relationship was established for the K+ channel blockers. The blockers elicited an unstable rhythmic activity, contrary to what occurred under control conditions. We also found that due to the specific changes that they elicit, the various blockers produce selective changes in the burst ratio. These results suggest that the various K+ channels contribute differently to the generation of locomotor activity.     

Calcium action potentials in innervated and denervated rat muscle fibres

We studied the generation of calcium action potentials (Ca APs) in innervated and denervated fibres of the extensor digitorum longus of the rat in a tetraethylammonium (TEA) sulphate solution plus 3–4 diaminopiridine (3–4 DAP). The main results are the following: (1) more than 90% of the innervated fibres were capable of developing well-sustained Ca APs that were blocked by Cd or nifedipine; (2) the incidence of Ca APs obtained from the denervated fibres was substantially lower than in the control preparations; (3) no relation was found between the appearance of Ca APs in the denervated fibres and the resting membrane potential (V _m), stimulus duration (500–2000 ms) or holding potential (–80, –100 mV); (4) The percentage of denervated fibres that exhibited Ca APs was increased significantly with the following procedures. First, by raising the external Ca concentration to 14 mM; second, by depleting the intracellular K concentration by overnight exposure of the muscles to a free K-Cs solution; (c) and third, by incubating the muscles in 500 nM apamin, a venom that inhibits the K conductance activated by Ca. Several factors may be involved in the lower incidence of Ca APs obtained in denervated fibres: (1) a diminished Ca current due to a reduction in the driving force as a result of an increment in the intracellular Ca concentration; (2) a persistence of a shunting K conductance that is not inhibited by TEA and 3–4 DAP; (3) a shift in the voltage dependence of the activation and inactivation parameters of the Ca current or the appearance of a new type of Ca channel with a different kinetics.     

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.