A study of the electrical basis for the inhibition and excitation in autonomically innervated smooth muscle

The aim of this thesis was to investigate the electrical and mechanical responses to inhibitory non-adrenergic noncholinergic (NANC) nerve stimulation in the bovine retractor penis muscle (BRP) and compare them with those to an inhibitory extract made from this muscle. The extract may contain the NANC inhibitory transmitter of the BRP and possibly of other smooth muscles. Because of species differences in the electrical response to NANC nerves in the rat and rabbit anococcygeus the effects of the extract on these tissues was also investigated. Prior to the investigation of the extract, both the excitatory and inhibitory responses to field stimulation in the BRP, and the effects of passive membrane potential displacement were studied using conventional intra- or extracellular (sucrose gap) recording techniques. The majority of cells in the BRP were electrically quiescent independent of the resting tone. The most frequent (in approximately 25% of preparations) form of spontaneous activity, oscillations in membrane potential and tone, may represent a pacemaker activity. The BRP had cable properties; the time constant and space constant indicated a high membrane resistance. In the absence of tone, field stimulation of the BRP evoked excitatory junction potentials (ejps) in every cell impaled and contractions, graded with the strength, frequency and number of pulses; spikes were not observed. Guanethidine (1-3 x 10-5M) abolished the ejps and contractions, confirming their adrenergic origin. Noradrenaline added exogenously depolarised and contracted the muscle. These effects were blocked by the a-adrenoceptor antagonists, phentolamine and prazosin. However, phentolamine (2.5x 10-6M) inhibited the contraction without reducing the ejp significantly. These effects may be independent of adrenoceptor blockade or the ejp may be mediated by a substance other than noradrenaline (e.g. ATP) released from adrenergic nerves. Prazosin (1.4 x lO-6M) failed to block either the ejp or contraction, indicating the possible existence of two types of adrenoceptor in the BRP; one activated by neuronally-released and the other by exogenously-added noradrenaline. ATP, a contaminant in the extract, also depolarised and contracted the BRP. Physostigmine reduced whilst atropine enhanced the ejps and contractions without similarly affecting the response to exogenous noradrenaline. This confirmed the presence of a cholinergic inhibitory innervation acting on the excitatory adrenergic fibres (Klinge and Sjostrand, 1977). TEA (1 x lO-4M) enhanced the ejp and contraction. Higher concentrations (0.5 to 10 x 10-3M) depolarised, increased the tone and evoked electrical and mechanical oscillations but no spikes. The depolarisation and contraction to exogenous noradrenaline were not enhanced, indicating that TEA acts on the adrenergic nerves. Some post-synaptic effect to block K+ channels also seems likely. The relationship between ejp amplitude and membrane potential in the double sucrose gap was linear and indicated a reversal potential more positive than -30mV. Electrotonic pulse amplitude decreased during the ejp, indicating an increased membrane conductance. Ejps and contractions were reduced following the replacement of the NaCl of the Krebs solution with sodium glutamate. This may be due to the effects of glutamate itself (e.g. Ca2+ chelation) rather than reduction in the membrane Cl- gradient. Tone usually developed spontaneously and was accompanied by membrane depolarisation (from -53 to -45mV) which may open voltage-dependent channels, causing Ca2+ entry and/or its release from intracellular binding sites. Field stimulation produced inhibitory potentials (ijps) and relaxations graded with the strength and number of pulses but showing little frequency dependence. Rebound depolarisation and contraction often followed the ijp and relaxation. Tetrodotoxin (3 x IO-6M), but not adrenergic or cholinergic antagonists, abolished the ijp and relaxation, confirming their non-adrenergic non-cholinergic neurogenic nature. The extract, prepared and acid-activated as described by Gillespie, Hunter and Martin (1981), hyperpolarised and relaxed the BRP, as did sodium nitroprusside and adenosine triphosphate (ATP). Unlike the activated extract or sodium nitroprusside, desensitisation to ATP occurred rapidly and without any change in the inhibitory electrical or mechanical responses to field stimulation. The ijp and relaxation in the BRP were insensitive to apamin but abolished by oxyhaemoglobin (4-8 x 10-6M), as were the responses to extract and sodium nitroprusside. In TEA (10-2M), field stimulation evoked relaxations with no accompanying electrical change. The ijp may be unconnected with or additional to another mechanism producing relaxation. The relationship between membrane potential and ijp in the BRP was non-linear. Ijp amplitude was initially increased during membrane potential displacement from -45mV to approximately -60mV. Thereafter (-60 to -l03mV) the ijp was reduced. Ijps were abolished at -27 and -103mV; reversal was not observed. The hyperpolarisation to extract was also enhanced during passive displacement of the membrane potential to more negative values (-57mV). Membrane resistance increased during the ijp. The extract produced inconsistent changes in membrane resistance, possibly because of the presence of more than one active component. K+ withdrawal failed to enhance the ijp or hyperpolarisation to extract and 20mM K+ did not abolish the the ijp at membrane potentials exceeding EK (-49mV). Thus, the ijp or hyperpolarisation to extract are unlikely to be mediated by an increased K+ conductance. Reducing the Cl- abolished the hyperpolarisation to field stimulation and extract. This occurred more quickly than the anticipated reduction in the Cl- gradient and may be due to Ca2+ chelation by the anion substitute (glutamate or benzenesulphonate) or blockade of the resting conductance which is normally inactivated by the transmitter. Ouabain (1-5x 10-5M), which reduces both the Na+ and Cl- gradients, abolished the ijp, implicating either of these ions as the ionic species involved. In the rat and rabbit anococcygeus, field stimulation and extract each reduced guanethidine-induced tone. This was unaccompanied in the majority of cells in the rat by any significant electrical response. In the remaining cells, inhibition of the membrane potential oscillations occurred. The rabbit anococcygeus differed in that inhibition of the electrical oscillations was observed in every cell exhibiting this behaviour. However, the majority of cells in the rabbit were electrically quiescent and showed only small hyperpolarisations to field stimulation and no electrical response to extract. Apamin (1 x 10-7M) failed to block the electrical and mechanical response to field stimulation in the rabbit but did inhibit transiently that to extract. The latter effect may be due to the initial excitatory effects of apamin. The similarities between the electrical effects of the extract and those of inhibitory nerve stimulation in the BRP, rat and rabbit anococcygeus muscles are generally consistent with their being mediated by the same active component. Moreover, the ijp in the BRP shows properties which have not been reported in other non-adrenergic noncholinergically innervated smooth muscles.

The structural and mechanical integrity of historic wood

Little is known about historic wood as it ages naturally. Instead, most studies focus on biological decay, as it is often assumed that wood remains otherwise stable with age. This PhD project was organised by Historic Scotland and the University of Glasgow to investigate the natural chemical and physical aging of wood. The natural aging of wood was a concern for Historic Scotland as traditional timber replacement is the standard form of repair used in wooden cultural heritage; replacing rotten timber with new timber of the same species. The project was set up to look at what differences could exist both chemically and physically between old and new wood, which could put unforeseen stress on the joint between them. Through Historic Scotland it was possible to work with genuine historic wood from two species, Oak and Scots pine, both from the 1500’s, rather than relying on artificial aging. Artificial aging of wood is still a debated topic, with consideration given to whether it is truly mimicking the aging process or just damaging the wood cells. The chemical stability of wood was investigated using Fourier-transform infrared (FTIR) microscopy, as well as wet chemistry methods including a test for soluble sugars from the possible breakdown of the wood polymers. The physical properties assessed included using a tensile testing machine to uncover possible differences in mechanical properties. An environmental chamber was used to test the reaction to moisture of wood of different ages, as moisture is the most damaging aspect of the environment to wooden cultural objects. The project uncovered several differences, both physical and chemical, between the modern and historic wood which could affect the success of traditional ‘like for like’ repairs. Both oak and pine lost acetyl groups, over historic time, from their hemicellulose polymers. This chemical reaction releases acetic acid, which had no effect on the historic oak but was associated with reduced stiffness in historic pine, probably due to degradation of the hemicellulose polymers by acid hydrolysis. The stiffness of historic oak and pine was also reduced by decay. Visible pest decay led to loss of wood density but there was evidence that fungal decay, extending beyond what was visible, degraded the S2 layer of the pine cell walls, reducing the stiffness of the wood by depleting the cellulose microfibrils most aligned with the grain. Fungal decay of polysaccharides in pine wood left behind sugars that attracted increased levels of moisture. The degradation of essential polymers in the wood structure due to age had different impacts on the two species of wood, and raised questions concerning both the mechanism of aging of wood and the ways in which traditional repairs are implemented, especially in Scots pine. These repairs need to be done with more care and precision, especially in choosing new timber to match the old. Within this project a quantitative method of measuring the microfibril angle (MFA) of wood using polarised Fourier transform infrared (FTIR) microscopy has been developed, allowing the MFA of both new and historic pine to be measured. This provides some of the information needed for a more specific match when selecting replacement timbers for historic buildings.

Examination of myocardial electrophysiology using novel panoramic optical mapping techniques

Optical mapping of voltage signals has revolutionised the field and study of cardiac electrophysiology by providing the means to visualise changes in electrical activity at a high temporal and spatial resolution from the cellular to the whole heart level under both normal and disease conditions. The aim of this thesis was to develop a novel method of panoramic optical mapping using a single camera and to study myocardial electrophysiology in isolated Langendorff-perfused rabbit hearts. First, proper procedures for selection, filtering and analysis of the optical data recorded from the panoramic optical mapping system were established. This work was followed by extensive characterisation of the electrical activity across the epicardial surface of the preparation investigating time and heart dependent effects. In an initial study, features of epicardial electrophysiology were examined as the temperature of the heart was reduced below physiological values. This manoeuvre was chosen to mimic the temperatures experienced during various levels of hypothermia in vivo, a condition known to promote arrhythmias. The facility for panoramic optical mapping allowed the extent of changes in conduction timing and pattern of ventricular activation and repolarisation to be assessed. In the main experimental section, changes in epicardial electrical activity were assessed under various pacing conditions in both normal hearts and in a rabbit model of chronic MI. In these experiments, there was significant changes in the pattern of electrical activation corresponding with the changes in pacing regime. These experiments demonstrated a negative correlation between activation time and APD, which was not maintained during ventricular pacing. This suggests that activation pattern is not the sole determinant of action potential duration in intact hearts. Lastly, a realistic 3D computational model of the rabbit left ventricle was developed to simulate the passive and active mechanical properties of the heart. The aim of this model was to infer further information from the experimental optical mapping studies. In future, it would be feasible to gain insight into the electrical and mechanical performance of the heart by simulating experimental pacing conditions in the model.