Ian W. Rodger

Muscarinic blockade of beta-adrenoceptor-stimulated adenylyl cyclase: the role of stimulatory and inhibitory guanine-nucleotide binding regulatory proteins (Gs and Gi)

1 The functional antagonism that exists between muscarinic and β‐adrenoceptor function in guinea‐pig tracheal smooth muscle was investigated by assessing Gs and Gi regulated adenylyl cyclase activity in isolated membranes. 2 Membranes from guinea‐pig tracheal smooth muscle contain both Giα and Gsα as assessed by Western blots with anti‐G‐protein antibodies. 3 GppNHp, a non‐hydrolysable analogue of guanosine 5′‐triphosphate (GTP), was shown to stimulate adenylyl cyclase activity at high concentrations (10−6−10−4 m). GppNHp also produced a concentration‐dependent reduction in pertussis toxin‐catalysed adenosine diphosphate (ADP)‐ribosylation of Giα. 4 Pretreatment of tracheal smooth muscle slices with methacholine (10−6 m) provoked a blockade of isoprenaline plus GTP, GppNHp‐ and GTP‐stimulated adenylyl cyclase. 5 Addition of methacholine to membranes did not trigger inhibition of GTP‐stimulated adenylyl cyclase activity but did block the isoprenaline‐mediated augmentation of GTP‐stimulated adenylyl cyclase activity. 6 Pretreatment of tracheal smooth muscle with methacholine (10−6 m) provoked a blockade of cholera toxin‐catalysed NAD+‐dependent ADP‐ribosylation of Gsα. 7 Phorbol‐12‐myristate 13‐acetate (PMA)‐treatment of tracheal smooth muscle slices actually enhanced GppNHp‐stimulated adenylyl cyclase activity in subsequently prepared membranes. 8 We suggest that methacholine in addition to inhibiting adenylyl cyclase via a Gi‐dependent mechanism induces a functional inactivation of Gs activity. These results together may explain the functional antagonism that exists between increased muscarinic tone and the ability of β‐adrenoceptor agonists to provoke excitation‐contraction uncoupling.

Forskolin, cyclic nucleotides and positive inotropism in isolated papillary muscles of the rabbit.

1 The effects of forskolin, isoprenaline, sodium nitroprusside and the frequency of stimulation were examined on cyclic nucleotide levels and tension responses in rabbit isolated right ventricular papillary muscles. 2 Increasing the frequency of stimulation from 0.01 Hz to 1.6 Hz induced positive inotropic responses that were not obviously related to alterations in the level of either cyclic AMP or cyclic GMP. 3 Isoprenaline induced rapid, concentration‐related positive inotropic responses that were associated with increases in the levels of both cyclic AMP and cyclic GMP. There existed good correlations between the increases in tension and the concentrations of both cyclic nucleotides measured in the tissues. 4 Forskolin induced concentration‐related positive inotropic responses that were slow to develop. These responses were accompanied by concentration‐related increases in the levels of cyclic AMP but not cyclic GMP. The tension responses correlated well with the levels of cyclic AMP measured. The cyclic AMP levels produced by forskolin were some 8 fold higher than those induced by isoprenaline for similar increases in tension. 5 Sodium nitroprusside was without inotropic effect either positive or negative; it nevertheless elevated cyclic GMP levels whilst slightly reducing cyclic AMP levels. 6 These data show that the ratio of cyclic AMP to cyclic GMP does not correlate well with changes in mammalian cardiac contractility. The data further suggest that whilst the intracellular concentration of cyclic AMP in rabbit ventricular myocardium may be an important determinant of positive inotropism, the relationship between the two parameters is more complex than simple proportionality between the tension generated and the amount of cyclic AMP measured within the cells.

Effects of phosphodiesterase inhibitors on normal and chemically‐skinned isolated airway smooth muscle

1 The effects of three phosphodiesterase inhibitors (papaverine, isobutyl methyl xanthine (IBMX) and SKF 94120) were examined on tension responses and cyclic nucleotide content (both cyclic AMP and cyclic GMP) of normal and Triton X‐100 skinned isolated trachealis of the guinea‐pig. 2 The three inhibitors were approximately equipotent in eliciting concentration‐dependent relaxation of histamine‐induced contractions of the trachealis. 3 Papaverine‐induced relaxation was associated with concentration‐related increases in the levels of both cyclic nucleotides. 4 IBMX at low concentrations (1 μmol l−1) produced significant relaxation (36%) of histamine‐contracted trachealis without changing cyclic nucleotide levels. At a ten fold higher concentration IBMX‐induced relaxation (95%) was associated with a selective increase in tissue cyclic GMP levels. Only at the highest concentration tested (100 μmol l−1) did IBMX increase cyclic AMP levels significantly. 5 SKF 94120 (1 μmol l−1) elicited a 23% relaxation of the contracted trachealis without altering the tissue content of either cyclic nucleotide. At the two higher concentrations tested (10 and 100 μmol l−1), SKF 94120‐induced relaxation was accompanied by a selective increase in the levels of cyclic AMP. 6 In the skinned trachealis Ca2+ (10 and 20 μmol l−1)‐induced contractions were significantly inhibited by the calmodulin antagonist calmidazolium (10 μmol l−1) and by cyclic AMP (10 μmol l−1), the catalytic subunit of cyclic AMP‐dependent protein kinase (0.1 μmol l−1) and cyclic GMP (10 μmol l−1). 7 Papaverine (100 μmol l−1) significantly inhibited (31 ± 6%) the Ca2+‐induced contractions of the skinned trachealis. Both IBMX and SKF 94120 were without effect. 8 It is concluded that cyclic nucleotide‐dependent mechanisms have an inhibitory action on the biochemical processes that lead to contraction of the guinea‐pig trachealis. The results suggest that a functional sarcoplasmic reticular and/or plasma membrane is essential for the expression of IBMX‐and SKF 94120‐induced relaxation. This is not the case for papaverine. The results also highlight the fact that significant relaxant responses of airway smooth muscle can be produced by phosphodiesterase‐inhibiting drugs without concomitant elevations in tissue cyclic nucleotide content.

Lack of effect of leukotriene D4 on Ca‐uptake in airway smooth muscle

1 The effects of verapamil on leukotriene D4 (LTD4)‐ and KCl‐induced contractions and 45Ca‐uptake were examined in guinea‐pig isolated tracheal smooth muscle. 2 Both LTD4 (0.1 to 200 nmol l−1) and KCl (8 to 125 mmol l−1) produced concentration‐dependent increases in tension in the tracheal preparations. 3 Verapamil (1 μmol l−1) inhibited the tension responses induced by both LTD4 and KCl. 4 LTD4 failed to increase the lanthanum‐resistant Ca content of tracheal smooth muscle at either low (EC25; 3 nmol l−1) or high (EC90; 50 nmol l−1) concentrations. Verapamil did not modify this result. 5 KCl (90 mmol l−1) increased the lanthanum‐resistant Ca content of the smooth muscle by approximately 60% over basal levels. This effect was completely inhibited by verapamil (1 μmol l1). 6 It is concluded that in this tissue, LTD4 utilizes principally an intracellular source of Ca2+ to initiate contraction whereas KCl is dependent upon the uptake of Ca2+ from the extracellular compartment. It is suggested that the inhibitory effects of verapamil may reflect an intracellular mechanism of action directed against Ca2+ release initiated by LTD4.

Pharmacological actions of a new β‐adrenoceptor agonist, MJ‐9184–1, in anaesthetized cats

1 The effects of (–)‐isoprenaline and the new β‐adrenoceptor agonist, MJ‐9184–1, on the lungs, on the cardiovascular system, and on slow contracting skeletal muscle have been compared in cats under chloralose anaesthesia. 2 Both amines reduced the increases in airways resistance produced by 5‐HT, depressed incomplete tetanic contractions of the soleus muscle, lowered the blood pressure and produced an increase in heart rate. In comparison with (‐)‐isoprenaline, MJ‐9184–1 had a long duration of action. 3 The effects of MJ‐9184–1 and (–)‐isoprenaline were antagonized by the β‐adrenoceptor antagonist, propranolol. 4 MJ‐9184–1 was approximately half as potent as (–)‐isoprenaline in its effects on pulmonary resistance and soleus muscle contractility, and one seventh as potent in producing chronotropic effects in the heart. 5 These results suggest that MJ‐9184–1 possesses some specificity as a β2 receptor stimulant.

Chronotropic and inotropic actions of amrinone, carbazeran and isobutylmethyl xanthine: role of phosphodiesterase inhibition

1 The chronotropic and inotropic effects of amrinone, carbazeran and 3‐isobutyl‐1‐methyl xanthine (IBMX) were examined in isolated preparations of papillary muscle and right atria from rabbit heart. The effects of the drugs on cardiac phosphodiesterase and cyclic nucleotide content were also examined. 2 Amrinone (2.4 × 10−4 M‐2 × 10−3 M), carbazeran (9.1 × 10−6 M‐1.2 × 10−3 M), and IBMX (1.8 × 10−5 M‐4.5 × 10−4 M) produced concentration‐dependent positive inotropic responses of papillary muscle preparations, the rank order of potency being carbazeran = IBMX > amrinone. Sub‐threshold positive inotropic concentrations of all three compounds potentiated the positive inotropic effects of isoprenaline; leftward shifts in the concentration‐effect curves were 5 fold (IBMX), 11 fold (amrinone) and 46 fold (carbazeran). 3 Amrinone and IBMX produced concentration‐dependent positive chronotropic responses in isolated right atria and showed a similar rate selectivity to isoprenaline, but carbazeran elicited a decrease in beating frequency. None of these drugs potentiated the positive chronotropic effects of isoprenaline. 4 Concentrations of amrinone, carbazeran and IBMX that produced similar positive inotropic responses were associated with different increases in papillary muscle cyclic AMP and cyclic GMP concentrations. 5 All three compounds inhibited right atrial and ventricular phosphodiesterase, with amrinone being the least potent. There was, however, a marked difference between the IC50 and EC50 values for phosphodiesterase inhibition and positive inotropy. In contrast the positive chronotropic effects of amrinone and IBMX were observed in the same concentration ranges that produced phosphodiestrease inhibition. 6 The results indicate that amrinone possesses a similar rate/force selectivity to isoprenaline and IBMX. In contrast, carbazeran exerts both positive inotropic and negative chronotropic effects. Phosphodiesterase inhibition and elevation of intracellular cyclic AMP concentration may be involved, at least in part, in the cardiac effects of these drugs.

The release of prostanoids during the acute pulmonary response to E. coli endotoxin in anaesthetized cats.

1 The administration of E. coli endotoxin (2 mg/kg i.v.) to anaesthetized cats results in a characteristic acute pulmonary response. This consists of increases in pulmonary artery pressure and airways resistance and a reduction in lung compliance. 2 Plasma concentrations of prostaglandin E2 (PGE2), PGF2α, thromboxane B2 and 6‐keto PGF1α were measured by radioimmunoassay in aortic and pulmonary arterial blood samples before, during and after the acute pulmonary response to endotoxin. 3 Endotoxin administration resulted in the rapid release of PGF2α and thromboxane B2 from the lungs. The time course of this release was parallel to that of the pulmonary hypertensive and airways responses to endotoxin. PGE2 and 6‐keto PGF1α were released more gradually and with greater variations between individual animals. 4 During the delayed shock phase (2 h after endotoxin) the concentrations of PGE2, PGF2α and 6‐keto PGF1α were once again elevated in both the aorta and pulmonary artery. Thromboxane B2 concentrations were not increased at this time. 5 These results suggest that PGF2α and thromboxane A2 may be mediators of the acute pulmonary responses to endotoxin.

Agonist-induced contractile responses of human bronchial muscle in vitro: Effects of Ca2+ removal, La3+ and PY108068

The effects of reducing the extracellular Ca2+ concentration ([Ca2+]0), the effects of La3+ and the dihydropyridine Ca2+ entry blocker PY108068 on contractile responses in human isolated bronchial strips have been compared. Reducing [Ca2+]0 or the presence of La3+ (1 mM) caused a reduction in basal tone, whereas PY108068 (1 microM) had no effect on unstimulated preparations. Response to KCl and A23187 were, in general, more markedly depressed by reducing the entry of extracellular Ca2+, with La3+ or PY108068 than were those to histamine, methacholine and LTD4. The effects of reducing [Ca2+]0, La3+ and PY108068 on responses to the receptor-mediated agents suggest that intracellular Ca2+ may be mobilized upon receptor activation, whereas responses to KCl and A23187 appear to be dependent upon the influx of extracellular Ca2+. More than one source of activator Ca2+ can therefore be involved in the development of contractions in human airway smooth muscle. These findings are in accord with those observed for in vitro airways preparations from experimental animals.

Effects of isoetharine on airways resistance, heart rate, and contractions of the soleus muscle of the anaesthetised cat.

Abstract The actions of the sympathomimetic bronchodilator, isoetharine, were compared with those of laevoisoprenaline, racemic isoprenaline and salbutamol, on the heart and lungs and on contractions of the soleus muscle of cats under chloralose anaesthesia. Isoetharine and salbutamol injected i.v. were approximately equipotent in all tests, and were about 8 times less potent than laevoisoprenaline both in decreasing the tension and degree of fusion of incomplete tetanic contractions of the cat soleus muscle, and in opposing the bronchoconstrictor action of 5-hydroxytryptamine. They were about 22 times less potent than laevoisoprenaline in increasing heart rate. In all tests, racemic isoprenaline was about 2 times less potent than laevoisoprenaline. The results suggest that the configuration of soleus β-receptors is closely similar to that of β-receptors in the lungs, and that the systemic administration of isoetharine might produce muscle tremor as a side-effect in man.

Effects of dantrolene sodium on respiratory and other muscles and on respiratory parameters in the anaesthetised cat.

Abstract The effects of intravenous dantrolene sodium on contractions of the tibialis anterior, the flexor digitorum longus, the soleus and the diaphragm muscles, and on respiratory parameters, blood pressure and heart rate were recorded in cats under chloralose anaesthesia. Dantrolene maximally depressed twitches of the tibialis anterior and flexor digitorum loncles to a significantly greater extent (ca. 90%) than it depressed those of the soleus and diaphragm muscles (ca. 72%). When the muscles were indirectly stimulated at frequencies high enough to produce complete fusion of responses, the depressant action of dantrolene was largely masked. The onset of this “break through” effect was evident with paired stimuli, and was maximal with trains of about 6 stimuli. Dantrolene was without effect on blood pressure and heart rate in all experiments; the slight effects that accompanied each injection were entirely attributable to the solvent (propylene glycol). In 8 out of 12 experiments, dantrolene was without any detectable effect on breathing. In one cat, however, which happened to be unusually deeply anaesthetised, dantrolene produced a decrease in the frequency of breathing and the animal tended to hold each breath for longer than usual. In 3 out of the 12 cats, dantrolene produced a slight increase in transpulmonary pressure, a slight reduction in tidal volume and a slight shortening of the duration of each respiratory cycle with no alteration in the frequency of breathing. It is proposed that the lack of effect of dantrolene on the respiratory parameters in most experiments was the result of a reflex increase in the frequency of discharge from the respiratory centre to the extent that resistant tetanic contractions of the respiratory muscles were evoked.