A comparison of the acute effects of radiation therapy, including or excluding the thymus, on the lymphocyte subpopulations of cancer patients.

Radiation therapy to either mediastinum or pelvis causes a rapid decrease in circulating lymphocytes of both B and T types and in addition an impairment in the function of the remaining lyphocytes, as measured by their ability to proliferate in response to mitogens. The acute depression is short-lived. Substantial recovery is apparent within 3 wk after cessation of therapy; however, most patients show a modest, chronic depression in both numbers and functional capacities of circulating lymphocytes. T cells are somewhat more sensitive than B cells, but both are affected. Irradiation of the thymus per se seems to have little influence on the acute changes which occur, as patients receiving pelvic and mediastinal (including thymic) radiotherapy show a similiar degree of lymphopenia and depression of lymphocyte responsiveness.

Electrical slow waves and tone of guinea-pig isolated trachealis muscle: effects of drugs and temperature changes.

1 Simultaneous recordings of electrical and mechanical activity have been made from guinea-pig isolated trachealis muscle. Electrical activity was recorded both by extracellular and intracellular techniques.2 Extracellular studies showed that the spontaneous development of tone was accompanied by electrical slow waves which frequently exhibited pronounced waxing and waning. Intracellular recording confirmed the discharge of these slow waves in individual cells. Extracellularly-recorded slow waves were often of greatest amplitude while the tissue was developing rather than maintaining tension. Some tissues became electrically quiescent on reaching peak tone.3 Cooling to 27.5 degrees C caused some relaxation. Slow wave amplitude and frequency fell, slow waves eventually being abolished. Subsequent rapid rewarming initially evoked a more profound relaxation. An intense discharge of slow waves then occurred as the tension rapidly rose again towards the pre-cooling value.4 Sodium nitrite, (-)-isoprenaline, adenosine and adenosine triphosphate (ATP) each evoked relaxation and reduced the frequency and amplitude of slow waves. High concentrations of these agents often abolished slow waves. The actions of these drugs were reversible.5 Treatment with methoxyverapamil (D600) 1 mumol/l for 15 min abolished slow wave activity but only evoked partial relaxation of the tissue.6 Acetylcholine, histamine and tetraethylammonium (TEA) each evoked contraction, but TEA was unique in consistently promoting slow waves and (in high concentration) spike activity. Spasm evoked by acetylcholine and histamine did not usually involve the initiation or promotion of slow waves. Indeed in appropriate concentration these two agents always suppressed slow wave activity. The actions of the spasmogens were reversible.7 It is concluded that the smooth muscle cells of the trachealis are electrically coupled. While co-ordinated slow wave activity is associated with the spontaneous development of tension in trachealis, it may not be necessary for the maintenance of the major part of the spontaneous tension exhibited by the tissue or for the spasm evoked by histamine or acetylcholine. Slow wave promotion by TEA suggests that the tissue may have a high resting potassium conductance which normally attenuates the slow waves. Slow waves may be suppressed by a variety of drugs acting by different mechanisms. Since D600 suppresses slow waves of the trachealis the mechanisms underlying the waves may be similar to those underlying spike activity in other smooth muscles.

Spasmolytic effects of cadmium and zinc ions upon the guinea-pig isolated ileum preparation.

1. An investigation has been made of the effects of cadmium and zinc ions upon the contractile response of the guinea-pig isolated ileum to methacholine, histamine, potassium ion and a.c. field stimulation. The metal ions depress the response to all of these agents.2. Radioisotope studies showed that cadmium and zinc ions have very much larger apparent volumes of distribution than sorbitol, both within whole ileum and within strips of the longitudinal smooth muscle layer. The results of these studies were indicative of surface binding and/or intracellular accumulation of these ions.3. It is suggested that cadmium and zinc ions directly depress smooth muscle contractility in a non-specific manner. This action may result from their binding to or accumulation within the muscle cells.

Evidence of poor conduction of muscle excitation in the longitudinal axis of guinea-pig isolated trachea.

1 Mechanical activity was recorded from one segment of guinea-pig trachealis muscle while intracellular electrical activity was simultaneously recorded from a contiguous segment. When the tissue was stimulated by 1-32 mmol/l tetraethylammonium (TEA) it became clear that the electrical and mechanical records were not directly correlated. 2 Dual recording of mechanical activity from two contiguous segments of trachealis revealed that one tissue segment could exhibit phasic activity whilst the other could exhibit tonic activity, despite exposure to the same concentration of TEA. 3 Histological studies revealed that the trachealis muscle was organized into bundles largely separated from one another by spaces filled with connective tissue. However, muscle bundles branched and formed anastomotic connections with near neighbours. 4 It is concluded that the cell to cell spread of muscle excitation is very poor along the longitudinal axis of the trachea. The trachealis muscle as a whole does not function as a single unit. Rather it may represent a series of effector units each comprising a small number of smooth muscle bundles.

Theophylline antagonizes some effects of purines in the intestine but not those of intramural inhibitory nerve stimulation.

1 Hyoscine- and guanethidine-treated preparations of longitudinal muscle of rabbit duodenum, guinea-pig taenia caeci and fundic strip relaxed when exposed to noradrenaline, adenosine, adenosine triphosphate (ATP) or to field stimulation of their intramural nerves. 2 In guinea-pig taenia caeci and fundus, theophylline 100 mumol/l had no effect on responses to noradrenaline, adenosine, ATP and intramural nerve stimulation. 3 In rabbit duodenum, theophylline 100 mumol/l antagonized some responses to adenosine but had no effect on responses to noradrenaline, ATP and intramural nerve stimulation. 4 Theophylline 1 mmol/l itself relaxed the intestinal tissues and in the fundic strip and taenia caeci, these relaxant effects were associated with abolition of spike activity and cellular hyperpolarization. In the taenia caeci, the amplitude of inhibitory post-junctional potentials was reduced. 5 Theophylline 1 mmol/l antagonized the twitch suppression produced by adenosine and ATP in the transmurally-stimulated guinea-pig ileum but not that evoked by noradrenaline. 6 It is concluded that theophylline can selectively antagonize some actions of purines in the intestine but that it does not specifically antagonize the effect of intramural inhibitory nerve stimulation.

Some effects of nifedipine in guinea-pig isolated trachealis.

In trachealis depolarized by a K+-rich medium, nifedipine (0.001-1 mumol 1(-1) caused concentration-dependent antagonism of CaCl2-induced increase in tension, moving the CaCl2 log concentration-effect curve to the right and depressing the maximal response. In trachealis in normal Krebs solution, similar concentrations of nifedipine had marked antispasmogenic activity against the responses to potassium chloride (KCl) and tetraethylammonium (TEA). However, nifedipine had little, if any, antispasmogenic activity against the responses to acetylcholine or histamine. Nifedipine 1 mumol 1(-1) was tested for spasmolytic activity in tissues generating tension in response to the EC50 of acetylcholine, KCl or CaCl2. In producing spasmolysis nifedipine was most effective against CaCl2 and least effective against acetylcholine. Nifedipine (0.01-1 mumol-1) had little or no effect on the tone of trachealis in normal Krebs solution. Intracellular electrophysiological recording showed that nifedipine 1 mumol 1(-1) could abolish spontaneous slow wave activity. This was associated with very minor depolarization and little or no loss of mechanical tone. In tissues treated with TEA (8 mmol 1(-1) nifedipine abolished spike and slow wave discharge and reduced mechanical activity to the pre-TEA level. It is concluded that nifedipine prevents KCl- or TEA-induced spasm by inhibition of Ca2+ influx. Spasm evoked by acetylcholine or histamine and the maintenance of spontaneous tone depend largely on mechanisms for increasing the cytoplasmic concentration of free Ca2+ which are resistant to nifedipine.

Some features of the spasmogenic actions of acetylcholine and histamine in guinea-pig isolated trachealis.

Intracellular electrophysiological recording showed that acetylcholine (1 mumol l-1) and histamine (2 mumol l-1) depolarized trachealis cells and often increased the frequency of slow waves. Higher concentrations of these agents caused greater depolarization and abolition of slow waves. Marked depolarization was often associated with the appearance of electrical 'noise'. These electrical phenomena were accompanied by tonic tension development in a contiguous segment of trachea. Electrical 'noise' and tension evoked by high concentrations of acetylcholine or histamine could be dissipated by washing the agonist from the tissue. Acetylcholine-induced 'noise' was resistant to tetrodotoxin (3 mumol l-1) and to hexamethonium (1 mmol l-1). Neither acetylcholine (10-1,000 mumol l-1) nor histamine (2-200 mumol l-1) increased the lanthanum-resistant calcium fraction of muscle-containing strips of trachea. It is concluded that, while developing tension under the influence of acetylcholine or histamine, trachealis cells depolarize markedly but there is relatively little cellular influx of Ca2+.

Antagonism of Ca2+ and other actions of verapamil in guinea-pig isolated trachealis.

In trachealis bathed by a K+-rich, Ca2+-free physiological salt solution, calcium chloride (CaCl2) at 0.01 to 10 mmol l-1 evoked concentration-dependent spasm. Verapamil (0.1 to 10 mumol l-1) was an effective antagonist of CaCl2. Spasm evoked by acetylcholine, histamine, potassium chloride (KCl) and tetraethylammonium (TEA) was studied in trachealis bathed by normal Krebs solution. Verapamil (0.1 to 10 mumol l-1) markedly suppressed spasm evoked by KCl and TEA. In contrast the actions of acetylcholine and histamine were much less affected by verapamil. Spasm evoked by prostaglandin E2 was studied in trachealis bathed by Krebs solution containing indomethacin (2.8 mumol l-1). Verapamil (0.1 to 10 mumol l-1) had little or no effect against prostaglandin E2-induced spasm. Verapamil (0.1 to 10 mumol l-1) had relatively little effect on the tone of trachealis bathed by normal Krebs solution. In contrast bathing in Krebs solution lacking CaCl2 caused almost complete tone loss. Extracellular electrophysiological recording showed that verapamil (10 mumol l-1) suppressed not only TEA-evoked spasm but also TEA-evoked slow waves and spike potentials. Verapamil also abolished the transient period of slow wave activity associated with the spasm evoked by KCl. Intracellular electrophysiological recording showed that TEA-induced spike activity was resistant to tetrodotoxin (3 mumol l-1). However, verapamil (10 mumol l-1) abolished the tetrodotoxin-resistant spikes without increasing the resting membrane potential. It is concluded that verapamil suppresses TEA- or KCl-induced spasm, slow waves or spikes by inhibition of Ca2+ influx. Spasm evoked by acetylcholine, histamine and prostaglandin E2 depends on mechanisms for increasing the cytoplasmic concentration of free Ca2+ which are resistant to verapamil. The failure of verapamil markedly to depress tissue tone is consistent with the proposal that tone results from the activity of endogenous prostaglandins.

A patch-clamp study of K(+)-channel activity in bovine isolated tracheal smooth muscle cells.

1. Single smooth muscle cells were isolated from bovine trachealis by enzymic digestion. The properties of large conductance plasmalemmal K(+)-channels in these cells were studied by the patch-clamp recording technique. 2. Recordings were made from inside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 10 microM. Large unitary currents of both Ca(2+)-dependent and -independent types were observed. Measured between + 20 and + 40 mV, the slope conductances of the channels carrying these currents were 249 +/- 18 pS and 268 +/- 14 pS respectively. 3. Lowering [K+] on the cytosolic side of the patches from 140 to 6 mM, shifted the reversal potentials of the two types of unitary current from approximately zero to much greater than + 40 mV, suggesting that both currents were carried by K(+)-channels. 4. The Ca(2+)-dependent and -independent K(+)-channels detected in inside-out plasmalemmal patches could also be distinguished on the basis of their sensitivity to inhibitors (tetraethylammonium (TEA), 1-10 mM; Cs+, 10 mM; Ba2+, 1-10 mM; quinidine, 100 microM) applied to the cytosolic surface of the patches. 5. Recordings were made from outside-out plasmalemmal patches when [K+] was symmetrically high (140 mM) and when [Ca2+] on the cytosolic side of the patch was varied from nominally zero to 1 microM. Ca(2+)-dependent unitary currents were observed and the slope conductance of the channel carrying these currents was 229 +/- 5 pS. 6. Activity of the Ca2+-dependent K+-channel detected in outside-out patches could be inhibited by application of TEA (1 mM), Cs+ (10mM), Ba2(+210mM) or quinidine (100 microM) to the external surface of the patch. 4-Aminopyridine (4-AP; 1 mM) was ineffective as an inhibitor. 7. The activity of the Ca2+-dependent K+-channel recorded from outside-out patches was reversibly inhibited by charybdotoxin (100 nM). 8. When whole-cell recording was performed, the application of a depolarizing voltage ramp evoked outward current which was dependent on the [Ca2 +] in the recording pipette and which could be reversibly inhibited by charybdotoxin (50 nM-I microM) applied to the external surface of the cell.9. We conclude that bovine trachealis cells are richly endowed with charybdotoxin-sensitive, large conductance, Ca2 +-dependent K+-channels. These channels carry most of the outward current evoked by a depolarizing ramp and could play a major role in determining the outward rectifying properties of the trachealis cells. The role of the large Ca2 + -independent K+ -channels remains unclear.

Cromakalim-induced relaxation of guinea-pig isolated trachealis: antagonism by glibenclamide and by phentolamine.

1. Tested against the spontaneous tone of guinea-pig isolated trachealis, cromakalim (0.1-100 microM), isoprenaline (1 nM-1 microM) and theophylline (1 microM-1 mM) each produced concentration-dependent relaxation. 2. Glibenclamide (0.1-10 microM) did not itself alter the spontaneous tone of the trachea nor did it modify the relaxant actions of isoprenaline or theophylline. In contrast, glibenclamide (0.1 and 1 microM) caused a concentration-dependent rightward shift of the log concentration-effect curve of cromakalim. Glibenclamide (10 microM) reduced the slope of the log concentration-effect curve of cromakalim and moved the foot of the curve back towards the control position. 3. Phentolamine (1, 10 and 100 microm) did not itself alter the spontaneous tone of the trachea nor did it modify the relaxant actions of isoprenaline or theophylline. In contrast phentolamine caused concentration-dependent depression of the log concentration-effect curve of cromakalim. 4. Neither prazosin (1 microM) nor yohimbine (10 microM) modified the spontaneous tone of the trachea. Prazosin and yohimbine each failed to antagonise the effects of cromakalim, isoprenaline and theophylline. 5. Intracellular electrophysiological recording showed that glibenclamide (1 microM) and phentolamine (100 microM) caused minor change in the resting membrane potential of trachealis cells. Slow wave activity was slightly depressed by these agents. In contrast tetraethylammonium (TEA; 8 mM) caused marked depolarisation, and promoted the conversion of slow waves into regenerative action potentials. These electrical changes were accompanied by tonic tension development. 6. Phentolamine (100 microM) and glibenclamide (1 microM) reduced and reversed both the relaxation and the hyperpolarisation induced by cromakalim (10 microM). 7. It is concluded that glibenclamide and phentolamine each provide selective antagonism of the relaxant action of cromakalim in guinea-pig trachealis. These agents also inhibit the plasmalemmal hyperpolarisation induced by cromakalim. The effect of phentolamine is unrelated to the blockade of alpha 1- or alpha 2-adrenoceptors. If either glibenclamide or phentolamine act to block the K+ channels opened by cromakalim, then such channels are not identical to those which endow the trachealis plasmalemma with its powerful rectifying behaviour.

The effects of the dihydropyridine Bay K 8644 in guinea-pig isolated trachealis.

In trachea bathed by Krebs solution containing indomethacin 0.8 mumol l-1, Bay K 8644 (0.01-1 mumol l-1) evoked mild spasm. Peak tension was achieved after 10 min and was generally less than 20% of an acetylcholine (ACh) maximum. The effect of Bay K 8644 was not potentiated by addition of 2.5 mmol l-1 potassium chloride (KCl) to the Krebs solution. Bay K 8644 (1 mumol l-1) caused a small potentiation of KCl and tetraethylammonium (TEA). In contrast it did not modify the actions of ACh or histamine. Bay K 8644 (1 mumol l-1) caused a small potentiation of the effect of calcium chloride (CaCl2) tested in trachea bathed by a K+-rich, Ca2+-free, MOPS-buffered physiological salt solution. Organic inhibitors of calcium influx such as nifedipine (0.1 mumol l-1), verapamil (1 mumol l-1) or diltiazem (10 mumol l-1) each caused marked depression of concentration-effect curves to KCl. Bay K 8644 (0.01-1 mumol l-1) provided concentration-dependent protection against this effect in all three cases. Estimation of calcium influx by the lanthanum technique revealed that Bay K 8644 (1 mumol l-1) was able to promote the cellular influx of Ca2+. Intracellular electrophysiological recording showed that Bay K 8644 (1 mumol l-1) caused no change in the resting membrane potential of trachealis cells and no change in the properties of the spontaneous electrical slow waves. However, Bay K 8644 was able to delay the slow wave suppression evoked by 1 mumol l-1 nifedipine. The ability of Bay K 8644 to promote Ca2+ influx and its ability to protect against the effects of several structurally-unrelated inhibitors of Ca2+ influx are consistent with Bay K 8644 acting as an agonist at the dihydropyridine receptor associated with the voltage-operated Ca2+ channel (VOC) of trachealis muscle. By this action it potentiates those spasmogens (KCl, TEA) which act by permitting Ca2+ influx through VOCs. In contrast it has no effect on those spasmogens (ACh, histamine) which principally act to liberate Ca2+ from intracellular sites of sequestration.

The effects of epithelium removal on the actions of cholinomimetic drugs in opened segments and perfused tubular preparations of guinea-pig trachea.

1. Isolated segments of guinea-pig trachea or perfused tracheal tubes were arranged for the recording of trachealis tension changes in Krebs solution containing indomethacin (2.8 microM). 2. In opened tracheal segments, epithelium removal caused modest (2-3 fold) potentiation of the effects of acetylcholine (ACh) and methacholine (MeCh) but failed to potentiate carbachol (CCh), bethanechol (BeCh), oxotremorine or KCl. 3. Pretreatment with ecothiopate potentiated effects of ACh and MeCh but not of CCh or BeCh. Removal of epithelium in ecothiopate-treated tissue potentiated effects of ACh and MeCh but not of CCh or BeCh. 4. Guinea-pig ileum challenged with ACh was used as a bioassay system for cholinesterase activity. Scrapings of tracheal epithelium did not hydrolyse ACh. 5. Histochemical staining revealed no fibres positive for acetylcholinesterase or pseudocholinesterase in the tracheal epithelium. However, the underlying tissues contained acetylcholinesterase-positive nerve fibres and the trachealis muscle itself stained positively for pseudocholinesterase activity. 6. Neither tetrodotoxin (3 microM) nor hexamethonium (500 microM) modified the ability of epithelium removal to potentiate ACh. 7. In perfused tracheal tubes where spasmogens were added to the luminal perfusate, epithelium removal potentiated effects of ACh (31 fold), CCh (10 fold), oxotremorine (2 fold) and KCl. 8. In perfused tracheal tubes where spasmogens were added to the Krebs solution superfusing the adventitial surface of the tissue, epithelium removal significantly reduced the potency of CCh, oxotremorine and KCl. 9. It is concluded that the selectivity and magnitude of the potentiation of cholinomimetics caused by epithelium removal depends on the route by which the cholinomimetic agent gains access to the trachealis muscle. The potentiation of acetic acid esters of choline seen in opened tracheal segments does not reflect the loss of epithelial cholinesterase activity and does not depend on the activity of nervous reflex arcs in the tracheal wall. The reduced potency of adventitially-applied cholinomimetics and KCl seen in epithelium-denuded tissue strongly suggests that the epithelium can moderate trachealis sensitivity to cholinomimetic agents not only by releasing epithelium-derived relaxing factor but also by acting as a barrier to drug diffusion.

Tracheal relaxation induced by potassium channel opening drugs: its antagonism by adrenergic neurone blocking agents.

1. We have studied the ability of some adrenergic neurone blocking agents to inhibit the tracheal relaxant actions of isoprenaline, theophylline and the potassium channel openers (KCOs) BRL 38227, pinacidil and RP 52891. 2. BRL 38227, isoprenaline, pinacidil, RP 52891 and theophylline each caused concentration-dependent suppression of the spontaneous tone of guinea-pig isolated trachealis. The maximal relaxant effects of isoprenaline and pinacidil were equal to that of theophylline. In contrast, the maximal effects of BRL 38227 and RP 52891 were approximately 85-95% of that of theophylline. 3. Guanethidine (5-500 microM) did not itself modify the spontaneous tone of the trachealis muscle but antagonized BRL 38227 in a concentration-dependent manner. Guanethidine (50 microM) also antagonized pinacidil and RP 52891. However, guanethidine did not antagonize either isoprenaline or theophylline. 4. Bretylium (50 microM) did not itself modify the spontaneous tone of the trachealis muscle but antagonized BRL 38227, pinacidil and RP 52891. Bretylium did not antagonize either isoprenaline or theophylline. 5. Guanidine (50 and 500 microM) did not itself modify the spontaneous tone of the trachea and failed to modify the tracheal relaxant activity both of BRL 38227 and theophylline. 6. BRL 38227 (1 and 10 microM) stimulated, in a concentration-dependent manner, the efflux of 86Rb+ from strips of bovine trachealis muscle that had been pre-loaded with the radiotracer. Guanethidine (50 microM), bretylium (50 microM) and debrisoquine (50 microM) did not themselves modify the efflux of 86Rb+ from bovine trachealis but each of these agents markedly inhibited the stimulant effect of BRL 38227 (10 microM) on 86Rb+ efflux.7. It is concluded that the adrenergic neurone blocking agents guanethidine and bretylium can inhibit the tracheal relaxant actions of KCOs such as BRL 38227, pinacidil and RP 52891 without antagonizing isoprenaline or theophylline. The ability of the adrenergic neurone blocking agents to antagonize BRL 38227 in promoting 86Rb+ efflux from trachealis muscle may suggest that the adrenergic neurone blocking agents act to prevent the opening of the plasmalemmal K+-channel that is involved in the tracheal relaxant actions of the KCOs.

The spasmogenic action of potassium chloride in guinea-pig trachealis.

Tissue bath experiments showed that potassium chloride (KC1) at 10-40 mmoll-1 evoked spasm of guinea-pig trachealis which was unaffected by atropine (1 mumoll-1), mepyramine (1 mumoll-1), tetrodotoxin (3 mumoll-1) or indomethacin (2.8 mumoll-1). Spasm evoked by KC1 was depressed in Ca2+-free Krebs solution or by exposure of tissues to LaCl3 (0.25-1 mmoll-1). Extracellular electrical recording showed that the spasm evoked by KCl 10 mmoll-1 was associated with promotion of electrical slow wave activity. Higher concentrations of KC1 abolished slow wave activity but caused further tension development. Intracellular recording confirmed the ability of KC1 10 mmoll-1 transiently to promote slow wave activity in individual trachealis cells. This action was associated with depolarization and tension development. Higher concentrations of KC1 evoked further tension development but slow waves were suppressed as the depolarization evoked by KC1 increased. KC1 (10-40 mmoll-1) increased the lanthanum-resistant calcium fraction of muscle-containing strips of trachea. It is concluded that KC1 acts directly on the smooth muscle of guinea-pig trachea. The spasmogenic action is associated with transient promotion of slow wave activity and a fall in resting membrane potential. The spasm involves the cellular influx of Ca2+ and is dependent on the presence of Ca2+ in the extracellular fluid.

Evidence that the spasmogenic action of tetraethylammonium in guinea-pig trachealis is both direct and dependent on the cellular influx of calcium ion.

1 Tetraethylammonium (TEA, 1-8 mmol/l) evoked spasm of guinea-pig trachealis which was unaffected by atropine (1 mumol/l), mepyramine (1 mumol/l) or tetrodotoxin (3 mumol/l). 2 The spasm evoked by TEA was markedly suppressed in Ca2+-free Krebs solution while that evoked by acetylcholine was much less affected. 3 Extracellular electrical recording showed that exposure to Ca2+-free Krebs solution suppressed both spontaneous electrical slow wave activity of the trachea and the spasm and slow waves induced by TEA. These effects were reversible. 4 TEA (2 and 8 mmol/l) increased the lanthanum-resistant calcium fraction of trachea. 5 It is concluded that TEA acts directly on the smooth muscle of guinea-pig trachea, that the spasm and electrical slow waves evoked are Ca2+-dependent and that the cellular influx of Ca2+ is increased.

A study of the sympathomimetic action of guanethidine on the isolated anococcygeus muscle of the rat.

Guanethidine, acting on the rat isolated anococcygeus, causes adrenergic neurone blockade (slowly terminated by washing), noradrenaline potentiation and, with higher concentrations, spasm (both rapidly terminated by washing). 2 The spasm is an indirect sympathomimetic action, for it is sensitive to phentolamine and reserpine and shows tachyphylaxis. 3 The concentration of cocaine equieffective with the spasmogenic concentration of guanethidine as an inhibitor of noradrenaline uptake caused much less spasm. Moreover, it did not enhance noradrenaline efflux from anococcygeus loaded with (-)-[3H]-noradrenaline, as guanethidine did. 4 The spasm induced by guanethidine in excess of cocaine is due to guanethidine-evoked noradrenaline release.

Inhibitory effects of AH 21-132 in guinea-pig isolated ileum and taenia caeci.

1. AH 21-132 is being investigated as a potential chemotherapeutic agent for bronchial asthma. The present experiments were designed to determine whether AH 21-132 shares the activity of theophylline as an antagonist at adenosine A1 receptors and to assess its potency as a relaxant in intestinal smooth muscle. 2. In the transmurally-stimulated guinea-pig ileum, theophylline (1 mM), but not AH 21-132 (1 and 10 microM), antagonized twitch depression induced by adenosine. Higher concentrations (100 microM and 1 mM) of AH 21-132 themselves had a depressant effect. Neither theophylline (1 mM) nor AH 21-132 (1 and 10 microM) antagonized twitch depression induced by noradrenaline. 3. AH 21-132 (100 microM and 1 mM) depressed maximum contractions of ileum induced by both acetylcholine (ACh) and histamine. 4. In ileum treated with hyoscine (1 microM), AH 21-132 (greater than 10 microM) caused a concentration-dependent depression of the log concentration-effect curve for potassium chloride. 5. Simultaneous extracellular electrophysiological and mechanical recording from taenia caeci showed that AH 21-132 (100 microM-1 mM) inhibited spontaneous tension waves and their associated bursts of electrical spike activity. 6. Intracellular electrophysiological recording from taenia caeci showed that the mechano-inhibitory effect of 1 mM AH 21-132 was accompanied by abolition of spontaneous spike activity. Following spike abolition, the membrane potential assumed a value very close to that observed during periods of electrical quiescence prior to drug exposure. 7. AH 21-132 inhibited the activity of cyclic AMP-dependent and cyclic GMP-dependent phosphodiesterases derived from homogenates of ileal smooth muscle. The effective concentration ranges were 0.1-1OOO microM and 1-1000 microM, respectively. Theophylline, too, inhibited these enzymes but in each case was less potent than AH 21-132. 8. It is concluded that AH 21-132 is devoid of antagonist activity at adenosine Al receptors which modulate ACh release from intramural cholinergic nerves in the ileum. At concentrations greater than IO microM, AH 21-132 has a relaxant effect on intestinal smooth muscle characterized by suppression of spontaneous action potentials but by minor change in resting membrane potential. AH 21-132 previously has been reported to depress the spontaneous tone of trachealis muscle with an EC50 value of less than lO microM and the present experiments therefore show that this agent is much less potent in inhibiting intestinal muscle. This potency difference cannot be attributed to a tissuerelated difference in the potency of AH 21-132 as an inhibitor of cyclic AMP- or cyclic GMPdependent phosphodiesterases.

Inhibitory responses to nicotine and transmural stimulation in hyoscine-treated guinea-pig isolated trachealis: an electrical and mechanical study.

Guinea-pig isolated trachealis muscle treated with hyoscine (1 microM) exhibited mechanical tone which could be suppressed by transmural stimulation and, in a concentration-dependent manner, by nicotine (10-1000 microM). Hexamethonium (500 microM) did not itself cause tone changes, antagonized effects of nicotine but did not antagonize those of isoprenaline. Tetrodotoxin (0.3 microM) did not itself cause tone changes, did not modify the action of isoprenaline but antagonized the effects of nicotine and very markedly reduced responses to transmural electrical stimulation. Guanethidine (50 microM) did not itself cause tone changes, potentiated the action of isoprenaline, antagonized effects of nicotine and reduced responses to transmural electrical stimulation. Propranolol (1 microM) did not itself cause tone changes, antagonized effects of both isoprenaline and nicotine and reduced responses to transmural electrical stimulation. Propranolol (10 microM) caused greater antagonism of isoprenaline but did not further antagonize nicotine or further reduce responses to electrical stimulation. Intracellular electrophysiological recording from hyoscine-treated trachealis showed that 10 microM nicotine caused little or no mechanical or electrical change. Higher concentrations (100 microM and 1 mM) evoked relaxation which was often though not invariably accompanied by transient hyperpolarization and transient inhibition of electrical slow waves in the impaled cell. Hexamethonium (500 microM), tetrodotoxin (0.3 microM), guanethidine (50 microM) and propranolol (1 microM) each suppressed the electrical or mechanical changes evoked by nicotine (100 microM). However, nicotine (1 mM) tested in the presence of propranolol (1 microM), caused relaxation which could be accompanied by slow wave suppression but not by change in resting membrane potential. Transmural stimulation of hyoscine-treated trachea with single pulses of supramaximal voltage and 0.5 ms duration evoked neither relaxation nor membrane potential changes. Stimulation with similar pulses in trains of 5 s duration evoked relaxation which was dependent on pulse frequency. In many cells this relaxation was not accompanied by membrane potential change. In other cells suppression of slow waves occurred. At high pulse frequencies (greater than 16 Hz) this was generally accompanied by membrane hyperpolarization. In tissue treated with hyoscine and propranolol (both 1 microM), transmural stimulation with pulse trains as described above always evoked relaxation but no membrane potential changes were observed. 10 It is concluded that nicotine and transmural stimulation can excite intramural noradrenergic nerves in guinea-pig trachea and thereby evoke relaxation. The membrane potential changes (slow wave suppression and hyperpolarization) are similar to those evoked by the administration of agonists at beta-adrenoceptors. Nicotine and transmural stimulation also excite non-adrenergic non-cholinergic inhibitory (NANCI) nerves. The relaxation evoked by the NANCI neurotransmitter is accompanied by little, if any, membrane potential change.

The effects of phorbol 12,13-diacetate on responses of guinea-pig isolated trachea to methylxanthines, isoprenaline and ryanodine.

1. Using guinea-pig isolated trachea, we have studied how phorbol 12,13-diacetate (PDA) modulates mechanical responses of the tissue to methylxanthines, isoprenaline and ryanodine. 2. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each inhibited the spontaneous tone of the trachea. Pretreatment with PDA (0.1-10 microM) converted relaxant responses to high concentrations of the methylxanthines into contractions. PDA produced no equivalent effect against isoprenaline. Pretreatment with verapamil (1 or 10 microM), nifedipine (0.1 microM) or incubation with Ca(2+)-free, EGTA (0.1 mM)-containing physiological salt solution (PSS) suppressed the contraction produced by caffeine or theophylline in PDA (5 microM)-treated tissues. 3. The ability of PDA (5 microM) to convert caffeine-induced relaxation into caffeine-induced contraction was retained in tissues pretreated with a combination of atropine (1 microM) and mepyramine (1 microM) and in tissues denuded of the airway epithelium. 4. Caffeine (10 microM-5 mM), theophylline (10 microM-5 mM) and isoprenaline (1 nM-1 microM), each relaxed trachea contracted with histamine (0.1 mM). The relaxation induced by caffeine, theophylline and isoprenaline was markedly reduced in the presence of PDA (5 microM) and the responses to high concentrations of caffeine and theophylline, but not those to isoprenaline, were reversed to contractions. Verapamil (10 microM) prevented the effects of PDA against caffeine- or theophylline-induced relaxation. 5. PDA (1 microM) enhanced the tracheal spasm produced by caffeine (10 mM) and theophylline (10 mM) in indomethacin (2.8 microM)-treated trachea maintained at 20 degrees C. This enhancement was reduced in the presence of verapamil (10 microM). 6. Tested in trachea bathed by K+-rich (40 mM), Ca2+-free PSS, CaCl2 (0.1-20 mM) caused concentration-dependent spasm. PDA (1-5 MicroM) did not significantly modify the shape or position of the log concentration-effect curve for CaCl2. In contrast, verapamil (1 and 10 MicroM) antagonized CaCl2.7. Tested in trachea bathed by indomethacin (2.8 MicroM)-containing PSS, ryanodine (1-100 MicroM) caused concentration-dependent spasm. PDA (5 MicroM) potentiated ryanodine. Verapamil (10 MicroM) inhibited ryanodine in inducing spasm and suppressed the ability of PDA to potentiate ryanodine.8. It is concluded that, in guinea-pig isolated trachea, PDA augments the spasmogenic activity of the methylxanthines and ryanodine. This effect of PDA does not result from PDA-induced suppression of spontaneous tone, from increased cellular entry of Ca2+ through L-type channels or from sensitization of the intracellular contractile machinery to activator Ca2+. The evidence suggests, instead, that PDA facilitates methylxanthine- or ryanodine-induced release of Ca2+ from the intracellular store.

The relaxant action of nicorandil in guinea-pig isolated trachealis.

Nicorandil (1-1000 mumol l-1) caused concentration-dependent relaxation of guinea-pig isolated trachealis. Propranolol (1 mumol l-1) did not modify the relaxant action of nicorandil but antagonized isoprenaline. Among K+-channel inhibitors tested, apamin (0.1 mumol l-1) and procaine (5 mmol l-1) did not modify the relaxant action of nicorandil. In contrast, tetraethylammonium (TEA, 8 mmol l-1) caused five fold antagonism. Trachealis exposed to K+-rich (120 mmol l-1) Krebs solution developed near-maximal tension. Nicorandil relaxed the K+-depolarized tissue though its concentration-effect curve was shifted markedly to the right. In tissues in which tone was induced by histamine, methylene blue (100 mumol l-1) antagonized nicorandil and sodium nitroprusside but did not modify the relaxant action of aminophylline. Intracellular electrophysiological recording showed that nicorandil (1 mumol l-1) could evoke some relaxation in the absence of electrical changes. Higher concentrations (10-1000 mumol l-1) reduced the amplitude and frequency of spontaneous electrical slow waves. Nicorandil also caused concentration-dependent hyperpolarization and relaxation. When the hyperpolarization was sufficiently pronounced slow wave activity was abolished. TEA (8 mmol l-1) induced slow waves which were surmounted by a spike potential. TEA slightly reduced the maximal hyperpolarization induced by nicorandil and increased the time required for nicorandil to abolish slow wave discharge. Procaine (5 mmol l-1) induced slow waves of relatively low frequency. Sometimes these were surmounted by a spike potential Procaine markedly reduced the hyperpolarization induced by nicorandil and increased the time required for abolition of slow waves. In studies of the efflux of 86Rb+ from muscle-rich strips of trachea, nicorandil (1000 mumol l-1) increased the efflux rate constant, whereas isoprenaline (1 mumol l-1) was without effect. It is concluded that nicorandil-induced relaxation does not involve the activation of beta-adrenoceptors but is partly attributable to the formation of nitric oxide from the nitrate moiety in its molecular structure. Nicorandil can evoke relaxation in the absence of membrane potential change but towards the upper end of its effective concentration range, nicorandil increases membrane K+ conductance and thereby evokes hyperpolarization of trachealis cells. The K+ channels opened by nicorandil are permeable to 86Rb, insensitive to apamin and TEA but may be inhibited by procaine.