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.

Antiplatelet therapy and clinical outcomes in cardiovascular diseases

Cardiovascular diseases (CVD) is a leading cause of death in the world. Despite effective treatment regimens for ischaemic heart disease (IHD) and ischaemic stroke, mortality and recurrence rates remain high. Antiplatelet therapy is on effective treatment and reduces the risk of recurrent heart attack and stroke. Nevertheless, there are patients who stopped or interrupted their antiplatelet therapy for certain reasons or some patients may be resistant or poor responders to antiplatelet therapy. Furthermore, there is evidence of rebound effect in platelet activity after antiplatelet cessation and this may associate with increased risk of cardiovascular event. This thesis is divided into five main chapters (chapters 3 to 7) which attempt to provide data to help resolve the uncertainty. Chapter 1 highlights the background of cardiovascular diseases and the global burden of cardiovascular and cerebrovascular diseases. The metabolism of platelets, antiplatelet therapy and current antiplatelet therapy guidelines are described, followed by discussion of the risk of cardiovascular event and changes in antiplatelet therapy. Chapter 2 describes the data source from Virtual International Stroke Trial Archive (VISTA) and National Health Service Greater Glasgow and Clyde (NHSGGC) Safe Haven, followed by definition of outcome measures. In chapter 3, Virtual International Stroke Trial Archive (VISTA) data was examined to test whether continue with the same antiplatelet therapy or changing to a new antiplatelet regimen reduces the risk of subsequent events in patients who experience a stroke whilst taking antiplatelet therapy. The findings indicate that subjects who switch to a new antiplatelet regimen after stroke did not have a lower early recurrence rate than subjects who continued with the same antiplatelet therapy. Observations on bleeding complications were similar in both groups. However, changing antiplatelet regimen after stroke was associated with more favourable functional outcome across a full scale modified Rankin Scale (mRS) at 90 days. In chapter 4, association between early or later initiation of antiplatelet with a recurrent ischaemic stroke and bleeding complications was assessed using VISTA data. The findings indicate that there was no association between a recurrent ischaemic stroke and timing of initiation of antiplatelet drug after stroke. However, early initiation was associated with increased risk of bleeding. In terms of functional outcomes, this study demonstrated that the mid-time and late initiation of antiplatelet therapy after acute stroke are associated with better functional outcomes compared with early initiation. In chapter 5, a nested case-control study was performed to explore the rate of antiplatelet cessation and interruption in a sample of patients with recent ischaemic stroke and to assess the risk of cardiovascular events associated with cessation and interruption of antiplatelet. It was found that there was no increased risk of cardiovascular event among patients who had early cessation or interrupted/stopped antiplatelet therapy within 90 days following acute ischaemic stroke. In chapter 6, the incidence and predictors of cardiovascular events after DAPT cessation were evaluated. The incidence of cardiovascular event while taking DAPT and following discontinuation of DAPT was 15.7% and 16.7% respectively. This study found that increasing age was associated with an increased risk of cardiovascular event, whereas, revascularization-treated patients and longer duration of DAPT, were each associated with a decreased risk. The duration of DAPT six months and less was associated a significantly higher risk for cardiovascular event. In chapter 7, an untargeted metabolomics analysis was performed while on DAPT (aspirin plus ticagrelor) and once they stopped ticagrelor to identify metabolite changes associated with cardiovascular events after stopping DAPT. Ten ACS patients were recruited in this study and data were analysed for seven patients. Three hundred eleven putative metabolites were identified. This study found 16 putative metabolites significantly altered following ticagrelor cessation. Of these, seven metabolites were from lipid pathway and down-regulated some up to 3-fold. On the other hand, adenosine, from nucleotide metabolism was upregulated up to 2.6-fold. It concluded that there are changes in numerous pathways following DAPT discontinuation and whether these changes differ in patients who have cardiovascular event after stopping DAPT warrant further investigation. In chapter 8, a summary of the findings of this thesis are presented as well as the future directions of research in this area.