This study has examined the expression and distribution of the type-1, type-2 and type-3 InsP<SUB>3</SUB>R subtypes in vascular smooth muscle in order to establish the roles of these subtypes. Immunoblotting of portal vein and aorta from neonatal (2-5 day old) and developed (6 week old) rat revealed comparatively greater expression of type-2 and type-3 InsP<SUB>3</SUB>R in the neonatal rat, compared to developed, and expression of type-1 InsP<SUB>3</SUB>R was decreased in neonatal rat compared to developed. In addition, there was a reorganisation of the internal calcium stores and altered intracellular InsP<SUB>3</SUB>R distribution observed between neonatal and developed rat. In permeabilised portal vein from developed rat, application of 100μM InsP<SUB>3</SUB> induced contractions of 54±7% of maximal activated contraction, whereas the permeabilised portal vein from neonatal rat, InsP<SUB>3</SUB> failed to induce contractility, indicating the type-1 InsP<SUB>3</SUB>R, but not type-2 and type-3 InsP<SUB>3</SUB>R subtypes are involved in excitation-contraction coupling. InsP<SUB>3</SUB>R subtype expression was also investigated in primary cultures of developed aortic cells. When seeded at subconfluent density these cells modulate to a "synthetic" phenotype believed to be similar to that found in vascular injury. InsP<SUB>3</SUB>R subtype expression is regulated during cell differentiation, and also in some vascular disease states. The type-2 and type-3 InsP<SUB>3</SUB>R subtypes may be involved in proliferating vascular smooth muscle, whereas the role of the type-1 InsP<SUB>3</SUB>R subtype is in excitation-contraction coupling in developed VSM. Therefore InsP<SUB>3</SUB>R expression may be a means of regulating the phenotypic properties of the vascular smooth muscle cell <I>in vivo</I>.

No description available.

No description available.

This thesis has studied aspects of biomineralisation, covering the inorganic mineral, the organic matrix, the possible use of phospholipid bilayer vesicles to control mineralisation, calcium and other metal binding to an antibiotic ionophore, Lasalocid-A and a study of a known inhibitor of biomineralisation, the glycopeptide antifreeze found in the plasma of fish which live under polar conditions. The mineral systems studied have been calcium carbonate formations in otoconia and otoliths, crystals which form part of the balance organs of the inner ear, and coccoliths, the earliest eukariotic formation of calcium carbonate, from an alga. Both these systems have been studied by ultra-high resolution electron microscopy with the observation that both types of structures grow in a unique manner, quite distinct from their geological counterparts; indeed the coccolith system involves two distinct mechanisms of growth for different parts of its structure, which is only 2 μm in diameter. Mechanisms of growth of both biominerals are proposed. The study of the organic matrix was less successful in that it was not possible to fully characterise an acidic matrix protein, but it has been shown that the soluble matrix consists of many polypeptide chains cross-linked together, which undergo a conformational change on dissolution from the insoluble matrix on which they lie in vivo and consequently give in vitro results which do not mimic the in vivo condition. Equally, the use of vesicles to control the formation of calcium carbonate was shown to be possible on occasion, but lipids are very unstable in the presence of calcium and no means of stabilising the system to produce consistent results was determined. Two studies were made by ¹H-nmr, the metal-ion complexes of the ionophore Lasalocid-A and the antifreeze glycopeptide of polar fish, in order to demonstrate principles of the handing of isolated ions and of crystallisation inhibition. In both cases, the biological action of the system was mimicked and followed by nmr and a mechanism for their function proposed.

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of impaired neuromuscular transmission. It is associated with small cell lung carcinoma (SCLC) in 60% of patients. There is considerable evidence that the defect in LEMS is caused by autoantibodies to voltage-gated calcium channels (VGCCs) on the nerve terminal. Two VGCC subtypes were demonstrated in cultured neuronal cell lines by the technique of K⁺-induced ⁴⁵Ca²⁺ influx: one subtype was inhibited by dihydropyridines (DHPs), the other by ω-conotoxin (ωCgTx). These may correspond to L and N channels previously described for neuronal tissue. The presence of these VGCC subtypes was confirmed by radioligand binding studies using [3 Hj-PN200-11 0 and 125 I-d)ωCgTx respectively. Pooled LEMS IgG inhibited K⁺-induced Ca²⁺ flux by 40% in SKNSH (human neuroblastoma) cells, while control IgG had no effect. The same LEMS pool reduced the density of ¹²⁵I-ωCgTx binding sites in SKNSH and MAR5 (human SCLC) cells by 57% and 43% respectively. These results provide further evidence that LEMS antibodies cause a loss of functional VGCCs. 78 LEMS and 88 control sera were tested for anti-VGCC antibodies by the precipitation of ¹²⁵I-ωCgTx-labelled VGCCs extracted from SKNSH cells. 42% of LEMS sera had significant levels of antibody (30-1466pM) compared to the healthy controls (<31pM). There was a high correlation between these results and those obtained using antigen extracted from MAR5 cells. Raised antibody titres (30-82pM) were also found among SCLC patients (47%) and patients with rheumatoid arthritis or systemic lupus erythematosus (56%). The incidence of positive sera was not significant among patients with other neurological disorders, including myasthenia gravis. Antibody titre did not correlate with disease severity across individuals. However, longitudinal studies in two LEMS patients showed an inverse relation between antibody titre and an electromyographic index of disease severity. Some of the antibodies detected may, therefore, be implicated in the neurological symptoms of LEMS. The assay may be a useful aid for the diagnosis of LEMS in some patients.

No description available.

Gypsum (calcium sulphate dihydrate) is of great industrial importance with over 95,000 ktonnes being used in the world per annum. The greatest use of gypsum is in the production of plaster (calcium sulphate hemihydrate) for use as an interior finisher. Plaster is produced by the calcination (thermal decomposition) of gypsum. The most popular design is a continuous calciner where gypsum is fed continuously and is directly heated by hot air. There are a number of different phenomena occurring within a calciner, including heat transfer, mass transfer, particle and gas mixing, elutriation and the dehydration reaction itself. All these processes interact with each other. Although a lot of research has been carried out in these areas already, the literature has been found to contain significant discrepancies. This study contains experimental work which has been carried out in order to better understand the physical processes occurring within a gypsum calciner. The rate of dehydration of gypsum (35-67μm in diameter) has been studied in a fluidised bed reactor. Experiments were carried out at bed temperatures of 100 to 170°C. The fluidising gas was air with water vapour pressures of 0.001 to 0.30 atm. The dehydrations were under differential conditions. The results show that the dehydration under these conditions can be successfully modelled using the two dimensional Avrami-Er'ovev expression. A study of the fluidisation and elutriation properties of gypsum in batch vessels (cylindrical and conical) has been carried out. The mechanics of elutriation has been investigated and modelled for various freeboards, superficial gas velocities and air humidities. Tracer tests have also been carried out on a laboratory scale continuous conical kettle. Sodium carbonate was used as the inert tracer material. Runs were carried out at different air and gas flowrates and different bed temperatures. Residence time distributions were elucidated. Finally, the above experimental data and component models have been investigated for their applicability to producing a model of the laboratory scale gypsum calciner.

A newly developed calcium sensitive liquid-liquid membrane electrode is used to analyse seawater off the Oregon coast in waters fed by the Columbia River runoff. For the analysis, an application of the method of standard additions is used requiring the assumption that the seawater is of so high a salt concentration that a small change in the overall ionic strength is insufficient to disturb the electrode response to calcium. Two equations describing the behavior of the electrode are treated and one is found applicable for use in seawater. Analyses by electrode are compared with analyses of the same water by atomic absorption spectroscopy with a degree of scatter in the correlation which is largely accounted for by 10% of calcium accuracy in the electrode readout device. Overall laboratory precision of the electrode and readout was 3% of calcium per standard deviation. The electrode appears to be much better for analytical purposes than ± 10% of calcium concentration. / Graduation date: 1968

The present research is focused on the topic of inflammation-drug interaction. Inflammation complicates many human diseases and conditions ranging from obesity to cancer. Therefore, the study of the effect of inflammation on drug pharmacokinetics and pharmacodynamics is pivotal. First, we tested the hypothesis that controlling inflammation using valsartan can restore the previously reported altered verapamil pharmacokinetics and pharmacodynamics. Such an effect is expected due to the anti-inflammatory properties of angiotensin II inhibition. Inflammation resulted in L-type calcium channel target protein (Cav1.2) downregulation and reduced verapamil potency in pre-adjuvant arthritis rat model. Valsartan treatment reversed the observed downregulation of L-type calcium channels thereby enhancing verapamil potency. This beneficial interaction, once proven in humans, may be of value in cardiac patients with superimposing inflammatory diseases. Second, we investigated whether the response to verapamil is reduced in experimentally induced acute myocardial injury (AMI) in rats. AMI caused a 75% reduction in verapamil potency and Cav1.2 target protein downregulation. If extrapolated to humans, our observations may suggest that L-type calcium channel downregulation can contribute, at least in part, to the poor outcome in myocardial infarction patients treated with calcium channel blockers (CCBs). Third, we studied the effect of obesity on the pharmacological response of CCBs in children with renal disease. Our data indicated that obese children are less responsive to CCBs than non-obese ones. Therefore, obesity should be considered when initiating antihypertensive drug therapy in children. Last, we were interested in finding out if the expression of other target genes is also altered by inflammation. We used real time polymerase chain reaction, after determination of the best housekeeping gene to be used as an internal control. Inflammation resulted in significant alterations of several molecular targets and transporters affecting the pharmacokinetics and pharmacodynamics of drugs. These findings may provide an insight into the effect of inflammation on drug targets and modulators of disease pathogenesis. In conclusion, inflammation is a missed ring in the chain of therapy. The research presented in this thesis will add to the inflammation-drug interaction field important findings that will help understanding the role of inflammation in pharmacotherapy outcomes. / pharmaceutical sciences

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