Chapter 4
4.1 Introduction
This chapter is divided in two parts. Part one looks at the inhibitory effects of a variety o f anti-allergic compounds in preventing histamine release from rat peritoneal mast cells stimulated by a - C T and other stimuli. Part two looks at the inhibitory effects of serine esterase substrates and other inhibitors in preventing histamine release from rat peritoneal mast cells stimulated by a-C T and other stimuli.
4 .1 .1 A n ti-A lle r g ic C o m p o u n d s
Mast cells play a key role in many allergic or immediate hypersensitivity type reactions through their high affinity IgE receptors. Owing to their ideal locations within mammalian bodies, they are able to interact to the highest possible degree with invading antigens. The widespread involvement of mastocytes in a diversity of allergic disorders has led to attempts to develop drugs that suppress mast cell function. The possible use of drugs which protect mast cells and prevent mediator release would therefore appear to be of great therapeutic benefit.
Through the years, a diverse range of drugs has been developed and prescribed to patients with allergic disorders. Many of these drugs are structurally unrelated and have different modes of action. The following sections look briefly at a range of anti allergic compounds and their ability to inhibit histamine from rat peritoneal mast cells stimulated by a-C T and other stimuli.
The introduction o f DSCG some twenty years ago provided a significant advance in the prophylactic treatment of asthma and other allergic conditions. DSCG is a derivative of the naturally occurring anti-spasmodic compound, khellin (a furano- chromone). Since its discovery, intense efforts have been made to elucidate the mode of action of the chromone. There have been many possible mechanisms o f action put forward. These include the possibility of DSCG exerting its effect on mast cells by stabilizing the cell membrane (48), by sequestering extracellular calcium ions or finally
by inhibiting cAMP phosphodiesterase (327,328). However, more recent studies have provided convincing evidence that the latter is unlikely to be the case. In particular, DSCG is a more potent inhibitor o f cyclic GMP- than cyclic AMP- selective phosphodiesterase (329). A more specific mode o f action o f DSCG was put forward by Theoharides and co-workers (182), who suggested that the effect o f DSCG on rat mast cells was associated with phosphorylation of a 78,000 dalton protein, which is thought to regulate the termination of the secretory response.
Nedocromil sodium is a recently introduced anti-asthmatic agent with structural similarities to DSCG and is thought to exert its effects via a similar mechanism (302).
Methyl xanthines such as theophylline and 3-isobutyl-l-methylxanthine (IBMX) are another group of drugs which prevent mediator release from stimulated mast cells by inducing an increase in cAMP levels.
The flavonoids are a group of naturally occurring compounds whose structure is based on 2-phenyl-4-chromone, otherwise known as flavone. One o f the most widely distributed flavonoids is quercetin, which occurs naturally as the glycoside quercitin (330). The first study of the action o f quercetin on histamine release from mast cells was made by Fewtrell and Gomperts (331). It was proposed that if quercetin could increase the efficiency of the Ca^'*’-ATPase responsible for maintaining low intracellular levels of calcium, it would be expected to inhibit histamine secretion, since this is dependent on a rise of intracellular free calcium.
p-agonists are potent inhibitors of mediator release from human lung mast cells (332). P2-agonists such as salbutamol interact with cell membrane associated p-
adrenoceptors, triggering off a series o f biochemical reactions culm inating in an increase in intracellular cAMP levels, which bring about the cessation of secretion.
M ost o f the compounds described are just a few examples o f drugs which prevent m ediator release from stim ulated mast cells. As insigh into mast cell biochemistry and its role in allergy is increasing, it is hoped that more effective and specific drugs may be developed which could prove to be therapeutically useful.
Chapter 4
4.1.2 Serine Esterase Inhibitors and other Inhibitors
One of the most remarkable attributes of enzymes is their specificity o f action. Only certain substrates are acted upon and a single type o f reaction takes place, without side reactions or by-products.
Another interesting feature o f enzymes is their enormous catalytic power. Enzymes are proteins, and thus relatively fragile molecules. They bring about their extraordinary catalytic effects in dilute aqueous solution at biological pH and moderate temperature, in sharp contrast to the rather extreme conditions often required to accelerate chemical reactions in organic synthesis.
One of first important studies on the specificity of enzymes was carried out by Fischer, who found that enzymes capable o f hydrolysing glycosides can distinguish between their stereoisomeric forms (333). In 1894 this observation led him to enunciate the principle that the substrate molecule fits the active site of the enzyme in a lock and key, or complementary relationship. Because chymotrypsin is secreted into the small intestine, it was first thought to be specific for the hydrolysis o f relatively long polypeptides formed by the action of pepsin on ingested proteins in the stomach, but later work revealed that chymotrypsin can also attack short peptides. The enzyme splits specific types of peptide linkages wherever they occur in a peptide chain. Moreover, chymotrypsin is specific for those peptide linkages in which the carbonyl function is contributed by arom atic am ino acid residues e.g. tyrosine, and phenylalanine. Chymotrypsin can also hydrolyze the esters of aromatic amino acids.
Tissue mast cells and blood basophils play a central role in the IgE-dependent immediate hypersensitivity reaction (334) and the process of degranulation of these cells after their activation has been widely studied (335,336). Results showed that the general inhibitor o f serine esterases, diisopropylfluorophosphate (DPP), inhibits histamine release from activated mast cells. Austen and Brocklehurst (335) showed that DPP at >5 mM completely inhibited the antigen-dependent release of histamine from chopped guinea pig lung but only when DPP was present at the time of challenge.
This indicates the involvement of the serine esterase following initiation of the secretory process by receptor bridging.
A chymytroypsin-type serine protease (chymase) has been purified from rat mast cells in several laboratories and also a trypsin-type serine protease (tryptase), with an associated protein (trypstatin) that inhibits the protease activity in the pH range above 7.5 (89-92,96). To clarify whether one o f these proteases, or some other putative protease closely linked with IgE-receptors (337), is involved in the process of mast cell degranulation, it was necessary to study the effect o f selective inhibitors and substrates o f serine esterases on histamine release from the activated mastocyte.