Hirst, Stuart John; (1990) A pharmacological study of protein kinase C activation by phorbol esters, in guinea-pig airways: Possible effects on the inhibitory non-adrenergic non-cholinergic system. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes the effect of phorbol esters, potent activators of the enzyme protein kinase C (PKC), on isolated guinea-pig respiratory tract tissues. Phorbol esters evoked qualitatively different responses in lung parenchymal and tracheal strips - contraction in the lung parenchyma and relaxation in the trachea. The basis of the relaxant effect was investigated. Relaxation occurred with the active 4β-isomer, but not the inactive 4β-isomer of phorbol dibutyrate (PDBu). The relative potencies of several active 4β-isomers, and the ability of cell permeant analogues of diacylglycerol (DAG, the endogenous PKC activator) to produce relaxation of the trachea, suggested that the response involved activation of PKC. Was the unexpected relaxation due to a direct or an indirect mechanism? The relaxation did not appear to result from down-regulation of responses to either histamine, carbachol or contractile prostanoids. In contrast, isoprenaline-induced relaxation was inhibited by 4β-PDBu, but not 4β-PDBu. Results with the relevant mediator antagonists/enzyme inhibitors indicated that the relaxation did not involve relaxant cyclo-oxygenase products, sympathetic neurotransmitters, β-adrenoceptor stimulants or naloxone-sensitive opioids, nor the action of histamine at H1- or H2-receptors, the action of adenosine at A1- or A2-receptors or the release of epithelium-derived inhibitory factors. The relaxation was also resistant to tetrodotoxin, but was potentiated by agents said to elevate tracheal cyclic AMP levels (eg. isoprenaline and aminophylline). One possibility which remained was that the relaxation was due to PKC-mediated release of the inhibitory non-adrenergic, non-cholinergic (NANCi) neurotransmitter. The extent of the relaxation in the trachea to 4β-PDBu seemed to correlate with the reported distribution of functional NANCi innervation, VIP-immunoreactive nerve fibres and VIP-receptors in the lung. Incubation of tracheal strips with the protease inhibitors, bacitracin and aprotinin, facilitated the response to NANCi nerve stimulation (by electrical field stimulation), exogenous VIP and to 4β-PDBu, suggesting a contribution of a protease-sensitive peptide to the relaxation. The voltage-dependent Na+ channel activators, veratridine (VT) and aconitine (AC), are reported to cause neurotransmitter release and to produce molecular changes in synaptoneurosomes and cortical slices consistent with generation of DAG (and thus activation of PKC). In the trachea these agents caused relaxation in the presence of atropine and propranolol; and the profile of relaxation to these alkaloids in tissues taken from lower down the respiratory tract was consistent with activation of NANCi mechanisms. Results obtained with inhibitors of DAG metabolism, R59022 and RHC80267, suggested that generation of DAG and its subsequent inactivation by the DAG lipase route could be involved in NANCi nerve-mediated relaxation, consistent with the hypothesis that PKC activation (as by phorbol esters) is implicated in the release of the NANCi neurotransmitter. Attempts were then made to differentiate neuronal and non-neuronal relaxant responses of the trachea to 4β-PDBu by (i) desensitising to NANCi nerve stimulation (using prolonged electrical stimulation and prolonged incubation with VIP or AC) or (ii) depleting neurotransmitters (with black widow spider venom or the purified toxin, olatrotoxin) or (iii) reduction of bathing fluid temperature. The general finding was that the relaxation produced by NANCi nerve stimulation, veratridine and 4β-PDBu all seemed to be inhibited by methods most likely to result in neurotransmitter depletion - in particular from NANCi nerves. However, an attempt to collect and concentrate the NANCi neurotransmitter released either by pharmacological or electrical stimulation failed, as did an attempt to assay for it using a cascade technique. In conclusion, it is proposed that the relaxation of guinea-pig trachea to 4β-PDBu occurs indirectly rather than by a direct action on the smooth muscle. While the results obtained do not prove that the relaxation is due to release of the NANCi neurotransmitter, most do appear to support it. However, other mechanisms for the relaxation have not been ruled out.

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