No description available.

No description available.

No description available.

No description available.

It is now believed that there is a significant association between skeletal blood flow and the movement of calcium and other ions in and out of bone. Bone is capable of regulation of this flow but this is affected by factors such as physiological stimuli, arterial blood pressure and humeral agents which can override this ability. The effects of vasoactive substances, noradrenaline and ATP and calcium regulating hormones, PGE2, PTH and calcitonin on bone blood flow and strontium clearance have been investigated in an experimental model in the rat. In order to proceed with this work smaller studies were undertaken to develop and validate the technique used to measure flow and clearance. These included;1. validation of choice of cannulation site,2. investigation of rat bone haematocrit,3. determination of an appropriate withdrawal rate. Cannulation of the carotid artery and insertion of the cannula into the ventricle provides adequate mixing of the microspheres, withdrawal at the caudal artery (0.197ml/min) an adequate reference sample. The haematocrit work suggests that a correction factor may be required when using this procedure as the haematocrit of rat bone is consistently less than that of the caudal artery. However the results in this study are too variable to incorporate this finding in the bone blood flow and clearance work. The main study involved the use of radioactive microspheres to estimate bone blood flow and strontium-85 for strontium clearance in rats weighing approximately 350 grammes. This involved cannulation of the carotid and caudal arteries with injection of the agent and microspheres vis the carotid and reference blood samples withdrawn from the caudal. For each animal blood flow and strontium clearance in bone, blood perfusion pressure and blood flow in muscle were measured and from these strontium extraction and vascular resistance in bone and muscle were calculated. Infusion of noradrenaline resulted in a significant decrease in blood flow and an increase in blood pressure, while ATP cuased a significant decrease in only blood pressure. Neither agent had any effect on strontium clearance. Parathyroid hormone produced a significant decrease in both strontium clearance and blood pressure with a general trend of decreasing blood flow with increasing concentration. While administration of PGE2 significantly reduced blood flow, pressure and strontium clearance. Calcitonin had no effect on any variable apart from strontium clearance. This was dependent on the degree of change at the 5OU. dose which represented an increase of 30% while at the other dose clearance remained relatively constant. With all groups, there was a significant relationship between blood flow and strontium clearance, but the slope of the regression was significantly different for PGE2 only. This shows that PGE2 was having a direct effect on bone independent of its effect on blood flow. In the normal untreated animal a change in flow is reflected by a change in clearance, indicating that some vascular mechanism is involved. The PTH, ATP, calcitonin and noradrenaline animals all demonstrate this same pattern suggesting that these also act through some vascular mechanism, either directly or indirectly. But PGE2 has a direct effect on strontium clearance suggesting indicating that some other mechanism may be involved, possibly through a non vascular effect. This would therefore invalidate the use of clearance measurements as estimtes of bone blood flow.

Uridine 5' triphosphate (UTP) is a pyrimidine nucleotide which is released from a variety of cells including platelets and endothelial cells. The release of UTP can lead to vasoconstriction if there is damage to the endothelial layer and hence its actions on the vasculature are important areas of investigation. The research presented in this thesis describes the measurement of isometric contractile responses of rat mesenteric arteries to UTP and of the electrophysiological measurements of ionic currents of smooth muscle cells isolated from these arteries. UTP induced contractions were recorded using various pharmacological tools to investigate the possible signaling mechanisms leading to the contractile response. Particular attention was given to the identification of the ion channels involved in generating the UTP induced contraction. It was clear that the contraction was dependent on Ca2+ influx and my results indicated that this influx was only partly due to an increased activity of voltage-gated Ca2+ channels and that non-selective cation channels were also important. The involvement of PLC is likely as using U73122, a PLC blocker, significantly reduced the UTP induced response. However, the potential involvement of PKC is less convincing as several PKC isoform peptide inhibitors, linked to the carrier peptide Tat(47-57) to render them membrane permeable, failed to affect the contraction. The ionic basis of the UTP induced contraction was studied by measuring the effect of UTP on various ionic currents measured in enzymatically isolated mesenteric artery smooth muscle cells using the whole-cell patch-clamp technique. UTP was found to inhibit both Kv and KATP currents, though in parallel to the contraction results, the UTP induced inhibition of these currents remained even in the presence of PKC block, suggesting a lack of involvement of PKC. Finally, application of UTP resulted in the activation of a non-selective current. An inhibition of K+ currents and the activation of a non-selective cation current would lead to membrane depolarization, the activation of voltage-gated Ca2+ channels and, dependent on the permeability of the non-selective channels, an additional route for Ca2+ influx and hence contraction.

The human Ether à-go-go Related Gene (hERG) channel makes up the pore forming subunit of the IKr channel. This channel is involved in the repolarisation of the cardiac action potential. Reduction in IKr may cause the prolongation of the action potential, leading to fatal arrhythmias. A large variety of potentially therapeutic compounds inhibit the IKr channels, leading to acquired long QT syndrome. Therefore characterisation of the channel and improvements to in silico models are needed to accurately predict potential hERG side effects. The aim of this project was to use a series of analogues to block the hERG K+ channel, using them as molecular rulers to measure the size of the inner cavity. The phenotype of block was examined to see whether increasing length, results in a change in the phenotype of block from a drug trapping type to a foot in the door one. This would have given an estimate of the size of the hERG inner cavity. Excised inside-out patches of hERG channels display a significant rundown with time after excision. The project also investigated the region of hERG responsible for rundown. A chimeric channel between hERG and bEAG, a closely related channel that does not display rundown, was produced. The aim was to produce a channel with hERG properties, but without the rundown characteristic. This chimeric channel could then be used in excised inside-out patch recordings. The series of derivatives were found to have high affinity to the hERG channel. However, they displayed unusual blocking characteristics, inhibiting the channel in the open state, yet unbound in the closed state. It was also found that the C-terminus of hERG appears to be responsible for the rundown characteristic. The exchange for that of bEAG resulted in a channel that had attenuated rundown.

No description available.

No description available.

No description available.