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Title: Inhibition and excitation in non-propulsive mammalian smooth muscle
Author: Baird, Agnes Anne
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1990
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Mechanisms underlying relaxation in response to inhibitory NANC nerve stimulation and putative neurotransmitters of these nerves have been examined in the guinea-pig internal anal sphincter (IAS) and compared with those in the bovine retractor penis muscle (ERP) and guinea-pig taenia caeci. Two types of techniques were employed. One which measured the effects of nerve stimulation and drugs on electrical membrane properties where intracellular microelectrode and simultaneous mechanical recording techniques were used. Drugs, for example ATP or cromakalim were applied by perfusion in the Krebs' solution, microinjection into the bath, or by hydrostatic pressure ejection. A second method assessed the underlying biochemical changes accompanying relaxation by measuring alterations in second messenger systems, for example cyclic AMP and cyclic GMP using radiomimmunoassay techniques. Electrical events were clearly an important accompaniment to mechanical inhibition in the IAS. Field stimulation (single pulse and 5 pulses at 5, 10 and 20 Hz; 0.5ms; supramaximal voltage) produced large inhibitory junction potentials of up to 15mV in amplitude which accompanied relaxation of 80% of muscle tone. Indeed, hyperpolarising electrotonic current passed into the IAS produced relaxation. The neurotransmitter which is released by field stimulation of the inhibitory nerves is probably ATP since exogenous application of purine by hydrostatic pressure ejection (5.8x10-4M; 10-55ms) produced a dose-dependent hyperpolarisation. The membrane potential change was similar in size, rate of decline and duration to the ijp. Neither hyperpolarisation nor relaxation could be achieved with the P2x-purinergic agonist, betaMeATP (10-5-10-3M) or the P2-purinergic agonist adenosine (10-3M) thus ATP was acting on the P2y-purinergic receptor. Inhibitory NANC neurotransmission was not peptidergic since VIP (10-7-10-5M), bradykinin (10-3M), neuropeptide Y (10-5M), bombesin (10-5M), leu-enkephalin (1.8x10-4M), met-enkephalin (1.8x10-5M), somatostatin (10-6-10-3M) and substance P (7.6x10-6 - 7.6x10-4M) each had no effect on the membrane potential of the IAS. There is also evidence that stimulation of -adrenoceptors by isoprenaline (10^-9 - 10^-5M) produced relaxation which was accompanied hyperpolarisation of the IAS. In all cases where hyperpolarisation and relaxation are associated in the IAS, the mechanism underlying the electrical change appeared to be an increase in K^+ conductance. Apamin (4.5 x 10^-6M) which blocks certain Ca^2+-mediated K^+ channels, antagonised the electrical and mechanical responses produced by field stimulation and ATP. Similarly, TEA (8x10^-2M), which blocks most K^+ channels, antagonised the hyperpolarisations and relaxations produced by field stimulation, ATP and isoprenaline. Indeed, the K^+ channel activator cromakalim (10^-9-10^-5M) produced hyperpolarisation and relaxation of the IAS suggesting that an increase in K^+ conductance is important in the mediation of mechanical inhibition of the IAS. Relaxation of the IAS was also produced without a significant change in membrane potential by altering the levels of cyclic nucleotides within the smooth muscle cells of the IAS. Forskolin (10^-9-10^-5M), which activates adenylate cyclase with a subsequent increase in cyclic AMP, relaxed the IAS. Similarly, sodium nitroprusside (10^-9-10^-4M) - a cyclic GMP phosphodiesterase inhibitor, and 8-bromo-cyclic GMP (10^-4M) each increased cyclic GMP and produced relaxation of the IAS. Direct measurement of cyclic nucleotide levels of the IAS showed that field stimulation (80 pulses at 8H_Z; 0.5ms; supramaximal voltage) and ATP (10^-4) elevated the cyclic AMP and cyclic GMP contents of the IAS. All other stimuli which produced slow, prolonged electrical and mechanical changes increased the level of only one cyclic nucleotide. Isoprenaline (10^-4M), cromakalim (10^-5M) and forskolin (10^-5) increased cyclic AMP content while sodium nitroprusside (10^-5M) increased the cyclic GMP content. Further investigation of other second messenger systems involved in relaxation of the IAS showed that increase in inositol phosphate turnover was not associated with stimulation of inhibitory P_2y-purinoceptors by ATP (10^-2M) in the IAS. However, an increase in inositol phosphate accumulation was produced by noradrenaline (10^-4M) and associated with contraction. A method was devised to measure the intraluminal pressure changes of the internal anal spincter in the anaesthetised guinea-pig using a Millar pressure transducer. Using this method the in vitro results were largely confirmed by this in vivo study. Basal intraluminal sphincter pressure was increased by noradrenaline acting on -adrenoceptors and decreased by isoprenaline acting on -adrenoceptors, ATP on P_2y-purinoceptors and 2-chloroadenosine on P_1-purinoceptors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology