A STUDY OF POTASSIUM (K+) RECTIFIER SYSTEMS IN THE CAT’S AIRWAY SMOOTH MUSCLE.
The effects of Amiloride and clibechamide on the0 Ach – induced contracted responses of the cat airway muscles were tested.
1. Ach (0.1m – 10ml) given cululatively caused a dose-dependent contraction of the cat airways smooth muscle.
2. Pre-treatment with glibenclamide (10m), which on its own caused a little transient contraction of the airways smooth muscle, potentiated the Ach-induced CCRC.
3. Pretreatment with amiloride (10um) caused an inhibition of Ach-induced CCRC.
4. While glibenclamide potentiated the, Ach-induced contraction, amiloride inhibits it showing that amiloride sensitive K+- channel caused a reversal of the glibenclamide effect.
5. These results indicated the presence of varying types of K+ – channels in the cat airways.
INTRODCUTION AND LITERATURE REVIEW:
1.1. BRIEF ANATOMY OF THE TRACHEAL SMOOTH MUSCLE:
The trachea is a mobile cartilaginous and membranous tube about 4-7cm long. It commences in the neck at the lower border fo the cricoid cartilage at the lev3el of the 6th cervical sertibra. It continues downwards in the middle and bifurvates, at the level between the 4th and 5th vertebrae, into left and right bronchi. It’s wall is a fibroelastic membrane whose patency is maintained by 15 – 20 ‘c’ – shaped rings of hyaline cartilage. The gaps lie posteriorly and are closed by a sheet of untreated muscle, the tracheal smooth muscle contains muscarinic receptors in addition to alpha adrenergic, beta – 2 adrenergic, H – I and H – 2 receptors (Range and Dale, 1988).
The tracheal smooth muscle is innervated predominantly by parasympathetic nerve supply via the recruitment laryngeal branch of the vagus. The vagus supplies sensory, secreto-motor and motor (trachealis muscle) fibres. There is no direct sympathetic nerve supply to the trachea, but it is believed that a sympathetic nerve supply to the traches, but it is believed that a sympathetic overflow from stimulation of bronchial and tracheal blood vessels will also stimulate the alpha – and beta receptors on the trachelis muscle (Rang and Dale, 1988).
1.2. THE PHYSICAL BASIC FOR SMOOTH MUSCLE CONTRACTION:
Smooth muscle is composed of fibres which are far smaller than those found skeletal muscle. The fibres are usually 2 – 5 microme in diameter and only 50 – 200 microns in length, in contrast to skeletal muscle fibres that are as much as long (Guytone, 1986).
Smooth muscle does not have the same striated arrangement of actin and myosin filaments as that found skeletal muscle. Micrographs any specific organisation in the smooth muscle cell that could account for contraction. However, recent special electron micrographic techniques suggest that the smooth muscle cell contains large numbers of actin filaments attached to so – called ‘’dense bodies’’, as illustrated in the figure below.
Figure1:1 arrangement of actin and myosin filaments in the smooth muscle cell.
Some of these bodies in turn are attached to the cell membrane whereas others are located throughout the cell but are held in place by a scaffold of structural protein cross-attachments from one dense body to another. Interspersed among the actin filaments are a few thick filaments assumed to be myosin filaments. However, there are only one-twelfth to one fifteenth as many of these ‘’myosin filaments’’ as actin filaments.
Despite the relative paucity of myosin filaments, it is assumed that they have sufficient cross-bridges to attract the many actin filaments and cause contraction by the sliding filament mechanism in essentially the same way that this occurs in skeletal muscle.
1.3 THE SLIDING – FILAMENT CONCEPT:
The first event in the contraction of smooth muscle is the increase in calcium ion concentration as a result an action potential or any other stimulus that causes calcium ion influx into the cell xytosol. Secondly, the calcium ions bind with calmodulin to form a calcium calmodullin complex. Thirdly, this complex in turn binds with or activates one of the light-chain polypeptides of the myosin head activating its ATPase activity. Fourthly, the newly excited ATPase activity of the head then causes cleavage of APT and other conformational changes of the mysin head leading to the ‘’walk-along’’ or sliding filament process.
As soon as the actin filament becomes activated by the calcium ions, it is believed that the heads of the cross-bridges from the myosin filaments immediately become attracted to the active sites of the actin filaments via an unknown mechanisms. It is postulated that when the head attaches to an active site, this attachment simultaneously causes profound changes in the intermolecular forces in the head and aim of the cross-bridge. The new alignment of the forces causes the head to tilt toward the arm and to drag the actin filament along with it. This tilt of the head of the cross-bridge is called the power stroke. Then immediately after tilting the head automatically breaks away from the active site and returns to its normally direction. In this position it combines with an active site further down along the actin filaments, then, a similar tilt takes place again to cause a new power stroke.
1.4 THE ROLE OF CALCIUM IN SMOOTH MUSCLE CONTRACTION:
One of the primary regulators of smooth muscle contraction is the sarcoplasmic calcium ion concentration and it s release is the foremost event preceeding smooth muscle contraction (Somlye, 1985). Precontraction values for cytorange of 10-7 to 10-6 molar but for contraction to take place these values rise to about 2 x 10-5m to 10-4m (Guyton, 1986).
Calcium ion mobilizing hormones or neurotransmitters stimulate the hydrolysis of phosphatetidylinositol 4, 5 – biphosphate generating two second messengers, inositol 1, 4, 5 – triphosphate (IP3), which releases calcium ions from non-mito-chondrial intracellular stores (strab et al, 1982; prentki et al, 1985; Gill et al, 1986) and 1, 2 – diacylglyceerol (DAG) which stimulates a calcium ion – activated, phospholipid-dependent enzyme, protein kidnase c (PKC) Baron et al, 1985; Nishisula, 1984; Rasmussen and Barett, 1984; somllyo, 1985; Berridge, 1987). In smooth muscles such as porcine coronary artery and guinea pig ileum, calocium ions released by IP3 has been proposed to contribute t o the formation of agonist induced contractions (Hashimoto et al, 1986; Watson et al, 1988).
It has been reported that agents which stimulate vascular smooth muscle e.g. angiohensin and epinephrine cause, during contraction consisting of an initial large transient response succeeded by a similar sustained response. Data obtained in other studies have given rise to the proposal that 2 calcium dependent processes are implicated in rapid crycling of cross-bridges resulting in force development, succeeded by a second process of force maintainance believed to be associated with latch bridgeds. This second process is reported to have a lower requirement for calcium than the first. Nishizuka (1984) suggested that during signal transduction there is synergism between the calcium ion pathway and the PKC pathway. In tissues with a maintained responses, the IP3 / calcium system is responsible for the initial transient response to an agonist and the DAG/PKC system for the sustained response (Rasmussen and Barrett, 1984; suematsu et al, 1984; Leitjen and Breeman, 1986).
Various physiological and pharmacological agent mobilize calcium and induce a change in the cytoplasmic free calcium concentration (ca2+)c. in tracheal smooth muscle, cholinergic stimulation is thought to cause an increase in (ca2+)c. both by stimulation of calcium ion inflix across the plasma membrane, and by the mobilization of calcium from intracellular stores, on the other hand, extracellular high potassium ion is though to cause a depolarization of the plasma ion is thought to cause a depolarization of the plasma membrane leading to a stimulation of calcium ion influx via voltage dependent calcium ion channels. The resulting changes in (ca2+)c. are though to evoke various bicchmical responses including phosphorylation of the twenty thousand (20,000) – da muosin light chain. However, the mechanism of the calcium dependent regulation of contraction is still not fullu understood. Studies on purified smooth muscle acto-myosin deonstrageed that phosphorylation of the 20.000 – Da myosin light chian kinase is required for the activation of myosin ATPase activity (Harthsorno and siemankiwski, 1981; kamm and stull, 1985; park and Rasemusen, 1986).
1.5. EVIDENCE IN SUPPORT OF THEE ROLE EXTRACELLULAR CALCIUM ION:
(A) Voltage – operated channel blockers such as verapamil, nifedipine and dialtizem reduce drastically agonist induced contractions (Goodman and Gilman, 1985).
(b) when extracellular calcium ion was reduced in the parenchyma of cat’s trachea, a reduction in Ach-induced contraction was observed (obtainime and dale, 1984).
1.6: EVIDENCE FOR THE MOBILIZATION OF INTRACELLULAR ca2+
(a) even after a decrease in the extracellular calcium ion concentration, spasmogens still produce contractions (obianime unpulblshied data).
(b) DMB – 8 dose – dependently inhibit spasmogen-induced contractions (obionime – unpublished data)
© computer – autoradiographic imagery substanctiate this evidence (obianime – unpublished data).
(d) ca2+ – mobilizing agents like carbamycin (A23187), ionomycin and vanadate dose – dependently stimulate contraction of the guinea pig airway smooth muscle muscle (obianime and dale, 1985).
1.7 EVIDENCE FOR IP3:
(a) there is evidence for PIP2 breakdown with the generation of IP3 and phosphateidc acid in smooth muscle.hashimoto et al (1985), using tracheal muscle with Ach as agonist; Bingam smith et al (1984), using cultured arterial cells with angio tensile as agonist; sekar et al (1984); Donaldson and Hill (1985), have also shown that PIP2 tunover does occur during stimulus activation coupling in guinea – pig ileal smooth muscles.
(b) there is evidence that IP3 is involved in increasing the calcium concentration and / or releasing stored calcium in skinned single muscle cells from poroine coronary artery; somyyo et al (1985), showed that IP3 caused calcium release and tension development in skimmed rabbit arterial muscle and has been postulated to be the chemical messenger in excitation contraction coupling in smooth an skeletal muscle (Baron et al, 1984; Vergara et al, 1985).
(c) there is evidence that IP3 is produced after ligand- receptors coupling and that this correlates with calcium efflux or increase in intracellular calcium ion concentration; doyle and Ruegg, (1985), measuring Ip3 and calcium feelux in a rat’s acrotic smooth muscle cell line, the ligand being vasopressin; Reynolds and Dubynals (1985), using noradrenaline in a smooth muscle cell line, the ligand being vasopressin; Reynolds and Dubyak (1985), using noradrenaline in a smooth muscle cell line and measuring IP3 level and also calcium increase with quin 2; kotligaff et al (1987), using histamine on cultured airway smooth muscle cell line, have also sown an increase in intracellular calcium concentration which is independent of extracellular calcium.
1.8 EVIDENCE FOR DAG/PKC”
a. Rasmussen et al (1984) observed that DAG caused the liberation of ca2+ by its action on PKC and also using PKC activators they found that PKC was involved in smooth muscle contraction.
b. Resemusen et al (1984); Danthaluri and Deth (1984) showed that the continuous presence of PKC activators, such as phorbol 12-myristate 13 – acetate (PMA) and mezeren, caused tonic sapsm in vascular smooth muscle of the art aorta and perfused rabbit ear artery.
c. protein Kinase c is in plentiful supply in smooth mscule (Minakuchi et al, 1981; Yu, 1981) and there is enough evidence that DAG activates PKC (Nishizuka, 1984).
d. the involvement of PKC/DAG in smooth muscle contraction is indicated by the fact that appropriate agonists activate specific types of muscle in vascular smooth muscle; carbachol in tracheal muscle) to cause PIP2 hydrolisis with the generation of IP3 and DAG; Grinendling et al (1984), in vascular smooth muscle; Takunwa et al (1986) in bovine tracheal muscle.
e. PKC has been shown to modulate the phospoirrylaiton and actin – activated activity of heavy meromyosin mediated by myosin light cahin kinase through the phosphorylation of Lc20 at different sites (Nishikawa et al, 1984).
1.9 ROLES OF POTASSIUM (K+)
Voltage – sensitive calcium cahnnesl which are affected by changes in cellular membrane potentials may play a major role in smooth muscle tone. Membrane potentials can be regulated by k+ channels. BRL 38227 and its (+) enantiomer, cromakalim, are of a novel class of compounds termed K+ channel openers that have been shown to produce relaxation of various visceral and vascular smooth muscles (Hamilton and western, 1989). Their main mode of action is due to an activation of k+ conductance resulting hyperpolarization of the resting membrane potential.
Several different potassium channels have been characterised in different tissues. They include a time-dependent, voltage – sensitive, delayed rectifier K+ channel (Beech and Bolton, 1989). Others are small (Okabe et al, 1990) and large (gelband et al, 1989) conductance ca2+ – activated k+ channels. Also, ATP – sensitive K+ channel (KATP) have been proposed (standen et al, 1989). kATP channels have been identified in vascular smooth muscle (Standen et al, 1989), cardiac muscle (Noma, 1983), skeletal muscle (spruce et al, 1985) and pancreatic B – cells (cook and hales, 1984). Particular interest his centred on the kATP channels due to their ability on activation to cause smooth muscle relaxation (Quast and cook, 1989).
It has been postulated that under normal physiological conditions the probability of kATP channels being in an open states is very low, since the intracellular concentration of ATP will be buffered by creatine phosphate at a level in excess of that required to close the channels (Dalvisc, 1990). Mcleod and piper (1992) worked in the guinera-pig isolated perfused heart using leukotrienes c4 and D4 and angtitensin II as constrictors and cromakalim s dilators. They found that ghlibenelamide (a selective kATP channel blocker) could restore peak leukotriene vasoconstriction affects which had earlier been maximally inhibited by cromakaim. The fact that glibenclamide ws able to produce this constrictor action indicated that osme of the kATP channels were in an open state. This suggests that, of the K+ channels present in the vasculature, the KATP channels in particular may be involved in the mainternance of resting tone of the vasculature within the isolated perfused heat. However, both low intracellular PH (Davies, 1990) and hypoxia with in the cotonary areries (Daut et al, 1990) have been reported to lead to an increase in the probability of open kATP channels. Both these situations could arise in the isolated perfused heart causing the probability of open kATP channels. Both these situations could arise in the isolated perfused heart causing the probability of open kATP channels to be higher than in the normal physiological state.
A spasmogen is an agent that is capable of indicing a contraction or spasm in a responsive tissue. In this series of experiments, the spasmogen used was Acetylcholine (Ach).
The contractile action exerted by Ach on the airway smooth muscle results from it (Ach) interaction with muscarinic receptos. It is now known that there are at least wo subolasses – M1 and M2 – of this receptor type (Hirschorvitz et al, 1984). However, present knowledge as regards the events associated with the activation of the receptor is relatively sparse. Inhibition of adenylate cyclase (Murad et al, 1962; Watanabe, 1983), enhanced permeability to monovalent cations (Burgen and spaero, 1968), accumulation of guanosine 3’, 5’- monophosphate (George et al, 1970), hydrolysis of phosphoinositides (Jafferji and Nichell, 1976), mobilization of, or increased intracellular ca2+ concentration (purtney, 1978; Bolton, 1981) have all been correlated with muscarinic stimulation. Most likely, the stimulation of muscarinic receptors by Ach is due to an interaction between several of the above mentioned process. The resultant effect of this stimulation depends, however, on the tissue and receptor type. Ach causes broncho-constriction through the mediation of m2 – type muscarinic receptors.
1.11, GLIBENCALMIDE (GLYBURIDE):
This is a potent oral hypoglycemic agent which is used mainly for non-insulin dependent diabetics. This is due to its ability to increase endogenous insulin release as well as improve its peripheral effectiveness (katzung, 1987). It belongs to the class of compounds referred to as sulfonylureas. It is metabolised in the liver and is contraindicated in patients with cardiovascular diseases, hepatio impairment, renal insufficiency and in elderly pateints.
In this series of experiments, however, we concerned ourselves only with the actions of this drug as it affects membrane potentials regulated by k+ channels. Glibenolamide appears to be a selective blocker of kATP channels (Sturgess et al, 1985; Belles et al, 1987; Quast and cook, 1989; standen et al, 1989).
This is a direct acting antikaliuretic agent. It inhibits, promptly, Na+ absorption and K+ erection (Na+/k+ exchange) at the distal cortical segment of the nephron. It is filtered at the glomeruli and secreted by the proximal convoluted tubules. This is essential to its activity, for it is effective only when presented to its distal site of action from the luminal side of the tubule.
Hyperkalaemia is likely to result following continued usage fo this potassium sparing diuretic.