The basis of beta-adrenergic bronchodilation.
B-adrenergic stimulants are indispensable for the treatment of bronchospasm and bronchial asthma. They have been in use for many years but little attention was paid to their cellular mechanism of action. Some new information on how tracheo-bronchial smooth muscle (TBSM) is relaxed by B-adrenergic agonists is reviewed in this paper. As a prelude, the structure and function of TBSM are also reviewed briefly.
The physiological role of TBSM is to adjust the calibre of the airways so that for any given demand for pulmonary ventilation, the dead space and the airway resistance, and therefore the work of breathing are maintained at an optimum level. This adjustment may go haywire due to one or more causes and bronchospasm is manifested. [Fig. 1] shows the structure of TBSM. The cells measure about 1 mm in length and about 4 u in diameter. It is a multi-unit type of smooth muscle in which each vagal cholinergic motor nerve fibre supplies one or two cells. There are no syncitial connections between the cells and no spontaneous activity (unlike intestinal smooth muscle). TBSM in vivo shows some neurally driven rhythmic activity which is unrelated to the respiratory rhythm but there is no peristaltic activity. The cell membrane is polarised and depolarised respectively in the relaxed and contracted states but there are no action potentials. Human TBSM is richly supplied by the vagus but there is little or no evidence of adrenergic innervtion. Natural sympathetic stimulation presumably depends only on circulating adrenaline and noradrenaline. Agonists which contract TBSM include acetylcholine, histamine, prostaglandin F2, 5-HT, SRS-A, bradykinin and K + . Relaxants include B-adrenergic drugs, theophylline, papaverine, khellin, muscarinic choline, histamine, prostaglandin F2,
Muscle may be regarded as an engine which converts biological fuel (ultimately in the form of adenosine triphosphate-ATP) into mechanical energy. The engine has regulatory and motive components which operate at body temperature unlike man-made engines. [Fig. 2] shows in a schematic form the relation of stimulus-response coupling' to ultra-structure, Ca+ + flux and biochemical processes in the smooth muscle cell. The shortening of the cell results from the cyclical formation and disruption of bridges between actin and myosin filaments. This process consumes energy in the form of ATP and is triggered by the interaction of Ca+ + with myosin. Thus [Ca + + ] i (the concentration of Calcium in the cytosol) finally determines the length and tension of muscle cells. Interaction of excitatory stimuli with TBSM, through various intermediate reactions, finally raises [Ca+ +] i, triggering a contraction. Energy is required for the reactions leading to the increase in [Ca+ + ] i as well as the shortening of the contractile protein (actomyosin).
The mechanisms described above are fundamentally the same in all forms of muscle. In the case of smooth muscle, however, one must remember that control is bidirectional and that excitatory as well as inhibitory stimuli operate peripherally upon the muscle. The rest of this review is devoted to some cellular aspects of inhibition (relaxation) of TBSM by B-adrenergic agonists.
Bioenergetics of Relaxation
Is chemically mediated relaxation an active process or a passive one? In other words does it involve running the engine (or some parts of it) in reverse or does it merely turn off the engine? It was reasonable to believe that relaxation by competitive antagonists of acetylcholine might involve only turning off the engine. However, in the case of B-adrenergic (and other) inhibitors the situation appeared to be more complex.
Energy usage in muscle may be assessed by the measurement of (1) work and heat output, (2) ATP content of the muscle or (3) oxygen consumption of the muscle. The first method is technically difficult and the data do not include the energy consumed by ion fluxes and chemical changes. The second method is very precise but it is a destructive, snapshot method; to reconstruct a dynamic sequential picture of energy changes in the tissue, a series of matched pieces of the same tissue under rigidly controlled conditions must be analysed. Oxygen consumption is a relatively slow, steady state method which is useful as a measure of average energy usage if oxygen is available throughout the tissue and glycolytic (and other anaerobic) energy pathways are held in abeyance. The absolute energy yield per unit of oxygen consumed is difficult to compute. Nevertheless, when changes in average energy usage in the same tissue have to be measured under different conditions, oxygen consumption is the simplest and most practical method.
Attempts to investigate the energetics of smooth muscle by measuring oxygen consumption were made by Bulbring. Unfortunately the results were equivocal due to the use of a spontaneously active muscle (guinea pig taenia coli) and adrenaline (both " and B agonist). The author designed and built an apparatus to investigate the problem using TBSM and isoprenaline. The apparatus consisted of a semi-closed micro-bath (0.6 ml) in which a thin piece of TBSM (15 mm long, < 0.7 mm thick) was suspended at optimal tension. A thermostatic system maintained the temperature at 37.5 + 0.005°C and the contents of the bath were stirred constantly by a coated iron fly rotated by a magnet revolving externally. A Clarke-type polarographic electrode measured the oxygen tension (content) of the bath fluid. The fluid could be renewed from a warmed, oxygenated reservoir. Oxygen saturation was over 95%, in fresh fluid and observations were completed before it fell to 90%. Using this technique, it was found that: (1) oxygen consumption of TBSM was proportional to the isometric tension during contraction produced by graded concentrations of acetylcholine or high K + fluid, (2) resting oxygen consumption was not affected by isoprenaline, (3) relaxation of TBSM by isoprenaline (in the presence of acetylcholine or high K + ) was not accompanied by any decrease in oxygen consumption and (4) relaxation by isoprenaline required extra energy equal to over half That required for a corresponding contraction..  Thus the answer to the question posed at the beginning of this section is: B-adrenergic relaxation is an active energy-consuming process. Apparently energy is required to reverse some cellular effect of the contractile agonist.
3',5'cyclic adenosine monophosphate (cAMP) has been claimed to be an "internal messenger" in many cells. In TBSM, it was found that dibutyryl cAMP (a permeable derivative of cAMP) failed to relax tissue activated by acetylcholine or high K + However, measurement of tissue cAMP by a radio labelled protein binding assay showed that its tissue concentration was elevated at the very beginning of B-adrenergic relaxation and this increase persisted throughout the relaxation.
Fate of Cell Calcium During B-Adrenergic Relaxation
As noted above, Ca ++ is an essential intracellular mediator of excitation-contraction coupling. A relationship between cell Ca and adrenergic relaxation of smooth muscle was recognised long ago by Schild. In the TBSM depolarized with high extracellular K + , isometric tension is proportional to [Ca+ +]o (extracellular concentration of Ca ++ ).4 Excess [Ca + +lo could overcome the relaxant action of isoprenaline probably due to a corresponding increase in [Ca ++ ] i. It follows that isoprenaline acts by lowering [Ca ++ ] i either by sequestration in intracellular organelles or by efflux from the cell. In either case, energy would be required.
The question remained whether isoprenaline acted by efflux or sequestration of cell Ca. Various techniques have been used to solve this problem in smooth muscles (not TBSM) with equivocal results and neither possibility could be ruled out. More recently it was suggested that isoprenaline acts by stimulating membrane Na-K ATPase and Na-Ca exchange. This hypothesis was investigated by the author and found to be untenable.
Methods to directly measure low levels of Ca in situ are now available. One of these is based upon the use of aequorin which is a Ca-sensitive photoprotein obtained from a luminous jelly fish; its purified preparations are available commercially. It is a non-toxic protein, active at physiological temperature and pH and it emits light when exposed to low concentrations of Ca ++ . Peak light emission is proportional to log [Ca ++ ] in the range 10-7 to 10-4 M. A method was devised by the author (unpublished work) to monitor with aequorin the fate of cell Ca during B-adrenergic relaxation of TBSM. Tissue was mounted in a reflective glass cuvette, thermostated and positioned in front of a photomultiplier inside a lightproof box. Isometric tension and aequorin-Ca luminescence in the oxygenated bath fluid could be recorded simultaneously. When normal fluid was replaced by low-Ca fluid, acetylcholine produced the usual response despite the near absence of external Ca. This was accompanied by a small increase in net Ca efflux (i.e. concentration of Ca ++ in the bath fluid). Addition of isoprenaline to cholinergically activated TBSM produced the expected relaxation without any increase in the net Ca efflux. Thus acetylcholine apparently acted by releasing Ca ++ from the intracellular stores and isoprenaline reversed this process without efflux of Ca from the cell.
The contraction of TBSM exposed to high K + -normal Ca++ fluid continues for some time when the fluid is changed to high K +-low Ca++ ; as the cell Ca gradually leaks out, the tension also falls slowly. When isoprenaline was added to the bath after changing from high K+ normal Ca ++ to high K+ -low Ca ++ fluid, the bath Ca ++ decreased significantly after 1 minute and more so after 2 minutes as compared to the paired controls. [Table - 1]. This finding suggests that the relaxation of TBSM by isoprenaline was brought about by the transfer of Ca from the contractile protein into cellular storage sites without net efflux of Ca from the cells.
The following conclusions may be drawn from the above account:
1. B-adrenergic agonists relax TBSM through energy consuming mechanism (s).
2. They act by lowering the concentration of intracellular ionised Ca and cAMP may mediate this effect partly or wholly.
3. The effect on cell Ca is sequestration within intracellular storage sites rather than efflux from the cell.