Typescript (photocopy) / 154 leaves, [15] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.) Dept. of Human Physiology, University of Adelaide, 1982

Thesis (Ph.D.) Dept. of Human Physiology, University of Adelaide, 1982. / Typescript (photocopy).

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "December 1997." Typescript. Vita. Includes bibliographical references (l. 176-196). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "December 1997" Typescript. Vita. Includes bibliographical references (leaves: 176-196). Also available on the Internet.

Thesis (Ph. D.)--University of Hong Kong, 1998. / Includes bibliographical references (leaf 133-171).

Thesis (doctoral)--Rijksuniversiteit te Groningen, 1980. / Summary and vita in Dutch. Highlights sheet in Dutch inserted. Vita. Includes bibliographical references (p. 125-140).

Calcium transport across the placenta is asymmetrical and is believed to be an active transport. An essential step in such a transport is translocation of the ion across a single plasma membrane. The objective of this thesis was to study the Ca2+ -related properties of the placental plasma membranes and to gain some knowledge of their role in Ca2+ -transport. Three Ca2+ -related properties were studied: 1. Ca2+ -binding to the placental plasma membranes; 2. The membrane bound enzyme Ca -ATPase; and 3. Ca2+ -uptake by the placental plasma membrane vesicles. Ca2+ -binding properties of the membrane preparation were studied by the use of a new method, the flow dialysis system. Two types of sites for Ca were found: 1) high affinity, low capacity sites, and 2) low affinity, high capacity sites. The high affinity sites had 10-fold higher affinity for Ca2+ than for Mg2+ . A calcium-stimulated, membrane-bound enzyme, namely Ca2+ -ATPase, was located in the placental plasma membranes. This enzyme is distinct from the Na+, K+-ATPase and alkaline phosphatase. The enzyme can be activated by Mg2+ but with lower efficiency. Both Ca2+ and Mg2+ activate the enzyme at the same site. A formula was derived, enabling one to predict very precisely the velocity of the enzyme incubated under any combination of Ca2+ and Mg2+ ; this relationship is presented in a three dimensional model. The formula can be used for other enzymes or other substrates, as was demonstrated with ATP and ADP. The placental plasma membrane vesicles are capable of accumulating Ca2+ . Ca2+ -uptake was defined as the amount of Ca2+ which is not available for rapid exchange and cannot be displaced by a high concentration of competitor in the presence of ATP. This definition is different from and more accurate than the one which is widely used and cited in the literature. An intravesicular Ca2+ concentration of 190 mM was recorded, which was 24-fold higher than the external Ca2+ concentration (8 mM). Ca2+ -uptake was dependent on ATP hydrolysis by the placental Ca2+ -ATPase. This process was independent of Mg2+ . It is suggested that while the substrate for Ca2+ -ATPase is Ca-ATP, the substrate for Ca2+ -uptake is Ca2+. The overall Ca2+ -related properties of the placental plasma membranes are independent of Mg and the entire process from binding to membrane through activation of the enzyme and finally Ca2+ -uptake is dependent on Ca2+ alone. This situation is unique to the placental plasma membranes. It is tempting to speculate that the link between the maternal and the fetal circulation is achieved by forming vesicles loaded with Ca2+ on the maternal side and unloading them through fusion with the basal plasma membrane on the fetal side. The Ca2+ -related properties of placental plasma membranes described in this thesis, provide many answers regarding the first step in the asymmetrical transplacental Ca2+ -transport. Further investigation is required before a full understanding of the entire process is achieved. / Medicine, Faculty of / Cellular and Physiological Sciences, Department of / Graduate

The excitation-contraction-relaxation cycle of the guinea pig ileum longitudinal smooth muscle was studied in muscles contracted by a muscarinic agent, cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD) and by 60 mM KC1. Aspects of the cycle were investigated by analyzing the active transport enzyme activities in the sarcolemma, the tissue Ca depots which could release Ca for contraction and the sensitivity of the contractile responses to extracellular ion changes. Essentially net changes of intracellular Ca, Mg, Na and K content during contractions were measured by a modified 'La method'. The tissues were washed for 30 min in 160 mM Tris-HCl solution (pH 7.4) containing 10 mM LaCl₃ at 4°C in order to seal the intracellular ions in the cell and displace extracellular ions. A method to loosen the 'intercellular cementing' substance by reducing the tissue Ca and Mg was developed as an adjunct to the preparation of a sarcolemmal enriched microsomal fraction. The method reduced the tenacity of the tissue and made the tissue easy to disrupt by a mild homogenizing procedure. The method also appeared to aid the extraction of contractile proteins. The microsomal fraction was not detectably contaminated by mitochondria and was enriched with vesicles of sarcolemma, probably originating from the muscle caveolae. The sarcolemma enriched microsomal fraction had a Ca-ATPase activity that was progressively stimulated by 10⁻⁷ to 2.4 x 10⁻⁴ M free Ca²⁺ , did not require Mg and was inhibited by La. The microsomal Ca-ATPase activity was not due to contamination by actomyosin. The actomyosin Ca-r-ATPase in the soluble fraction had a higher affinity than the microsomal Ca-ATPase for Ca and for La. The microsomal Ca-ATPase activity was postulated to be associated with an active Ca pump thought to he located in the cayeolae. The microsomal fraction had a Mg-dependent ATPase that could Be stimulated by Na, but K and ouabain had very little additional effect. The addition of an activating factor in the soluble fraction conferred some K and ouabain sensitivity to the Mg-dependent Na-ATPase, which indicated that a Na,K-ATPase was present in this tissue. Low doses of ouabain contracted the longitudinal ileum but the responses were not antagonized by raising the external K concentration five fold, as would be expected if ouabain acted by inhibiting the Na,K-ATPase. However, the ouabain response was rapidly lost when extracellular Ca was removed from the medium and the decline of the response followed the same time course as the loss of extracellular Ca. The peak of the ouabain contraction coincided with significant increases of intracellular Ca and Na, but K loss was not apparent until relaxation ensued. The results suggested that ouabain has an early direct effect on membrane permeability before it inhibited the Na,K-ATPase. CD (2 x 10⁻⁷ M) and 60 mM KCl induced phasic and tonic contractions of the longitudinal muscle of the ileum. The phasic contraction declined from 100% to 7% over 10 min when Ca was omitted from the physiological medium. This decline followed the time course of the loss of extracellular Ca. This, together with the fact that low concentrations of LaCl₃ inhibited the phasic component, indicated that Ca bound to the outer aspect of the cell was responsible for the phasic component. The tonic component was lost more rapidly than the phasic component when the Ca was removed from the Tyrode's solution. The tonic component seemed activated by free Ca mobilized from the extracellular space. The extracellular origin of the Ca for contraction was consistent with the observed small net gain of intracellular Ca that occurred during the phasic and tonic contractions. The minimal volume of the sarcoplasmic reticulum and the abundance of caveolae was also consistent with the high sensitivity of the tissue to extracellular Ca concentrations. The intracellular Ca gained during contraction wa,s extruded within 30 sec after the CD or 60 mM KCl were washed out of the tissue bath, Following washout of CD, the muscle was quiescent for the 20 to 30 min 'equilibration' phase. Spontaneous activity was absent during this phase and tension was below baseline. After a maximal CD contraction, a second response to CD or to 60 mM KCl induced during the 'equilibration' phase had an altered or desensitized biphasic appearance. Responses of the muscle to CD for 10 min were accompanied by a cytoplasmic loss of K. After washout of CD, the K was regained slowly over 20 to 30 min. Stimulation of the tissue by 60 mM KCl did not cause a loss of K from the muscle nor did it cause desensitization of the muscle. Higher extracellular K concentrations decreased the time required after CD contractions for the return of spontaneous activity and prevented muscle desensitization to repeated doses of CD, probably by accelerating the return of intracellular K levels to normal. It was proposed that during contraction, elevated intracellular Ca activated K channels, thereby increasing K permeability and causing the 'after-hyperpolarization' and subsequent desensitization which follows muscarinic induced contractions. / Pharmaceutical Sciences, Faculty of / Graduate

This thesis is a study of the effect of lipid injections on complement titers of guinea pigs.

No description available.