The present studies were undertaken to determine the involvement of protein kinase C (PKC) in the regulation of rat granulosa cell steroidogenesis in vitro. The effects of PKC activators (1-oleoyl-2-acetylglycerol (OAG); 1,2-dioctanoylglycerol (DiC$\sb8$) and phorbol 12-myristate 13-acetate (TPA)) and inhibitors (DL-Sphingosine (ESP) and 1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine free base (H$\sb7)\rbrack$ on basal and FSH-, (Bu)$\sb2$cAMP-, forskolin- and calcium ionophore A23187-stimulated pregnenolone (P$\sb5$), progesterone (P) and 20$\alpha$-hydroxy-pregn-4-en-3-one (20$\alpha$-OH-P) secretion by granulosa cells were studied. OAG, when continually present in the culture medium (MEM), significantly stimulated P$\sb5$, P and 20$\alpha$-OH-P secretion during 6 to 24 h culture periods. It also markedly increased the conversion of exogenous P$\sb5$ to P and 20$\alpha$-OH-P and exogenous P to 20$\alpha$-OH-P during 24 h cultures. Pretreatment of granulosa cells with TPA for 1 h or treatment for up to 6 h resulted in a significant increase in P$\sb5$, P and 20$\alpha$-OH-P secretion. Except for 20$\alpha$-OH-P production, which was stimulated by the phorbol ester during all culture periods studied, secretion of P$\sb5$ and P (in the presence or absence of exogenous hormones and the inhibitors of steroidogenic enzymes) were substantially inhibited by TPA during a 24 h incubation. However, when granulosa cells were incubated with both OAG (20 $\mu$g/ml) and TPA (40 ng/ml), progestin secretion was increased irrespective of the duration of incubation. PKC inhibitors dose-dependently suppressed the stimulatory effect of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) with complete inhibition noted at 100 $\mu$M of H$\sb7$ and 10 $\mu$M of ESP. Diacylglycerols and TPA exerted divergent effects on FSH-, (Bu)$\sb2$cAMP- and forskolin-stimulated progestin secretion. FSH-stimulated accumulation of P$\sb5$ throughout the culture periods (1-24 h) was markedly increased by OAG (20 $\mu$g/ml) but inhibited by TPA (40 ng/ml). OAG (5-80 $\mu$g/ml) and DiC$\sb8$ (20 $\mu$g/ml) significantly enhanced FSH-induced progestin secretion during 6 h and 24 h culture periods and increased steroid synthesis in 24 h cultures in the presence of (Bu)$\sb2$cAMP or forskolin. In contrast, TPA significantly inhibited FSH- and (Bu)$\sb2$cAMP-stimulated progestin secretion during both 6 h and 24 h of incubation. Pretreatment of granulosa cells with TPA (40 ng/ml) for 20 h to down-regulate PKC, decreased progestin secretion during subsequent incubation with FSH (150 ng/ml) and prevented any stimulation by OAG (20 $\mu$g/ml). The effects of OAG (20 $\mu$g/ml) and TPA (40 ng/ml) on FSH-induced steroid secretion appeared to be additive when both PKC activators were present together and differed significantly from those when OAG and TPA were present with FSH separately. Diolein (a nonpermeable diacylglycerol), 4$\alpha$-phorbol 12,13-didecanoate and phorbol 13-monoacetate (two phorbol esters with no tumor promoting activity) did not influence basal or FSH-stimulated steroid secretion by granulosa cells. (Abstract shortened by UMI.)

Previous studies from our laboratory, using a modified loop microperfusion technique (microstop-flow conductivity) in vivo, have shown that NaCl transport by the thick ascending limb (TAL) is impaired in potassium depleted (K-DEP) rats. The degree of impairment in TAL NaCl transport is highly correlated with plasma potassium (K$\sp+$) concentration and is completely reversed when extracellular fluid potassium concentration (ECF (K$\sp+$)) is increased by acute potassium infusion. These experiments assessed NaCl transport by the TAL in the absence of axial flow by measuring the conductivity of tubular fluid emerging from an early distal tubule site after different intervals of stop-flow (10-60 seconds) in perfused nephrons. Since these measurements of TAL transport were made in the absence of axial flow, the significance of the defect in NaCl transport in K-DEP rats could not be determined under in vivo conditions of physiological flow and chloride (Cl$\sp-$) delivery. To determine the quantitative importance of this impairment in NaCl transport by the TAL, Cl$\sp-$ reabsorption was measured in functionally isolated perfused single loops of Henle in vivo, using the technique of continuous microperfusion. Cl$\sp-$ reabsorption was measured in loop segments microperfused at 22 nL/minute using a modified perfusate which minimized proximal nephron transport. This modification of the loop perfusate allowed the measurement of furosemide-sensitive Cl$\sp-$ reabsorption in the perfused loop segments such that net Cl$\sp-$ uptake in this study can be attributed primarily to carrier-mediated Cl$\sp-$ transport by the TAL. Others have provided evidence that TAL NaCl reabsorption is aldosterone dependent in adrenalectomized animals. In our rats given a K-free diet, both plasma (K$\sp+$) and plasma (aldosterone) were significantly reduced. Since aldosterone release is regulated by ECF (K$\sp+$), the purpose of the present study was to determine whether an aldosterone deficiency and/or reduced ECF (K$\sp+$) mediates inhibition of TAL Cl$\sp-$ transport in potassium depletion. Using the described microperfusion conditions, it was possible to show that the defect in TAL Cl$\sp-$ reabsorption in K-DEP rats was quantitatively significant and can be rapidly reversed by the acute systemic infusion of potassium. Acute administration of aldosterone, in the presence of sustained hypokalemia, failed to reverse the impairment in TAL Cl$\sp-$ reabsorption in K-DEP rats. However, the acute infusion of potassium, in the presence of an aldosterone antagonist, in K-DEP rats rapidly reversed the defect in TAL Cl$\sp-$ reabsorption to control levels. Additional studies showed that despite normalization of ECF (K$\sp+$) by acute potassium infusion in K-DEP rats, aldosterone levels failed to increase to control levels within this time period. This is the first study to demonstrate that the restoration of plasma (K$\sp+$) in K-DEP rats is not immediately associated with a parallel rise in plasma (aldosterone). As well, new knowledge was obtained from the present studies which showed that although a minimal (aldosterone) is required for normal TAL Cl$\sp-$ transport to occur, this steroid hormone does not regulate Cl$\sp-$ reabsorption by this nephron segment. Therefore, these results provide conclusive evidence that in K-DEP rats the rapid reversal of defective TAL Cl$\sp-$ reabsorption seen with acute potassium infusion occurs via an aldosterone independent mechanism.

The properties of type IIX fibres in mouse skeletal muscle and the factors affecting the expression of myosin heavy chain (MHC) in these fibres were studied. Type IIX fibres were recognized on the basis of a lack of staining with antibodies directed against type I, IIA and IIB MHC. The IIX MHC isoform contained a determinant common to all type II MHCs, but lacked epitopes specific for types IIA and IIB MHCs, as well as an epitope that was present in all other MHCs. Fibres expressing IIX MHC accounted for about one-third of the total fibre number in mouse fast-twitch muscles. Sciatic nerve crush with subsequent reinnervation resulted in the formation of "type IIX fibre groups" in tibialis anterior and gastrocnemius, suggesting that the IIX phenotype is neurally regulated. The association of specific MHC isoforms with individual fibre types was confirmed by gel electrophoresis. Using 5%, 6% and 3-5% gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) IIX MHC was found to co-migrate with IIA MHC. Immunohistochemistry and gel electrophoresis of single fibres from the superficial part of the tibialis anterior muscle (TAS) showed the existence of hybrid fibres co-expressing IIB and IIX MHC. Myosin light chain (MLC) analysis of IIX fibres revealed the presence in these fibres of MLC 3f in amounts significantly smaller than that contained in IIB fibre types which suggests that type IIX fibres have a lower Vo than type IIB fibres. Physiological, histochemical and morphometrical properties of fast-twitch single motor units were studied. Single motor units were functionally isolated by microdissection of the ventral root, the glycogen depletion technique was used to demonstrate the muscle fibres in the motor unit and monoclonal antibodies were used to identify their MHC composition in order to correlate physiological, histochemical and morphometrical studies. These studies revealed that IIX motor units had contractile properties similar to those of types IIA and IIB motor units but had morphological, physiological and biochemical properties that distinguish them from the latter two types. IIX motor units had a resistance to fatigue, cross-sectional fibre area and motor unit area intermediate between IIA and IIB motor units. Finally, the effect of altered thyroid hormone status and age on the expression of IIX MHC was studied. Hypothyroidism led to a decrease in the expression of IIB MHC and an increase of IIX MHC in TAS muscle, while hyperthyroidism had no significant effect. The proportion of type IIX and IIB fibres in TAS muscle of the mouse undergoes specific age-related changes that can not be detected with conventional histochemical techniques. Specifically, the proportion of fibres expressing IIB MHC decreased and the proportion of fibres expressing type IIX and co-expressing type IIB and IIX MHC increased with age. It is proposed that fibres co-expressing IIX and IIB MHC represent a transitional fibre type involved in an age-related transformation process.

This thesis examines the morphological and physiological changes that occur in the gills of the rainbow trout, Oncorhynchus mykiss, as a result of chloride cell proliferation. In the first component of this thesis (Chapter 2), the effects of chloride cell hyperplasia and hypertrophy on ion transport capability and the morphological diffusing capacity of the gill were evaluated. These experiments were performed to test the hypothesis that chloride cell proliferation benefits ionic regulation at the expense of efficient gas transfer. Concomitant with the surface morphology results, significant increases in Na$\sp+$ (J$\rm\sb{in}Na\sp+)$ and Cl$\sp-$ uptake (J$\rm\sb{in}Cl\sp-)$ were observed following treatment with the individual combined hormone regimens. Conversely, the gas morphological diffusion capacity, as indicated by the morphological parameters measured, of hormone treated fish was reduced, and was inversely correlated to chloride cell fractional surface area. Surface morphometric analysis showed that the hormone treatment increased the average chloride cell surface area by 2.7 x and chloride cell density by 2.2 x; the combined effect was a five-fold increase in chloride cell fractional area. While the P$\rm\sb aO\sb2$ values of hormone-treated and control fish were similar at P$\rm\sb wO\sb2>12.0$ kPa, the arterial O$\sb2$ tensions of treated fish were significantly lower than those of the control group for P$\rm\sb wO\sb2\leq12.0$ kPa. In comparison, to control fish at all environmental O$\sb2$ tensions, the hormone-treated fish exhibited elevated P$\rm\sb aCO\sb2$ values and a significant acidosis. The effects of chloride cell proliferation on blood gas parameters in hormone-treated fish were accompanied by significantly elevated ventilation amplitudes and lowered ventilation frequency. (Abstract shortened by UMI.)

ABSTRACT HAS BEEN ARCHIVED 10/23/96

Atrial muscle stretch is widely believed to be the main stimulus for ANF release. However, we demonstrate in this study that although stretch induces an immediate increase in ANF output, this release rapidly decays even though hormone stores are not significantly depleted. In the present work, this phenomenon was studied in an isolated rat atria preparation using double isotope labelling. The tissue was labelled with $\rm\sp C$-leucine for 3 h followed by a 1 h chase, and then with $\rm\sp3H$-leucine for 1 h. A final 1 h chase period was conducted with the tissue under basal (0.2 g load) or stretched (5 g load) conditions. During this final chase period, the $\rm\sp C$-ANF represented ""older", stored ANF and the $\rm\sp3H$-ANF the "newly synthesized" peptide. Following both the $\rm\sp C$- and $\rm\sp3H$-leucine pulses, immunoprecipitable (IP) isotope incorporated into ANF appeared in the chase medium within the first 10 min and stabilized to lower levels after 20 min of chase. Stretch resulted in an immediate significant increase in immunoreactive (ir) ANF release and a decrease in the medium $\rm\sp C$-ANF specific activity (S.A.). However, no change was observed in the medium $\rm\sp3H$-ANF S.A. but the tissue S.A. tended to decrease. It is concluded that, in the basal state, a portion of ANF is immediately and preferentially released upon synthesis, while the remainder is presumably taken up into tissue stores and released from them at a lower rate. The secretory response to stretch was demonstrated to consist of a rapid, short-lived burst of newly synthesized ANF, suggesting an increased translocation of newly synthesized hormone into a stretch-sensitive, rapidly depletable pool. Given the nature of this pool, additional factors yet to be characterized likely come into play to maintain chronically elevated circulating levels of ANF.

The ocular artifacts derive from the potential difference between the cornea and the fundus of the eye. This can be represented by an equivalent dipole with its positive pole directed toward the cornea. The DC potential between the cornea and the forehead measures approximately +13 mV. The scalp-distribution of the ocular artifacts can be described in terms of propagation factors--the percentage of the EOG present at the EEG electrodes. These factors are significantly different for blinks and upward eye-movements. The source dipoles for blinks and saccades are different--blink dipoles point radially whereas saccade dipoles point tangentially, in the direction of the eye movement. Blink and eye movement potentials are generated by different mechanisms--blink potentials are generated by the eyelid sliding over the cornea, eye movement potentials by the rotation of the ocular dipole. A very small downward rotation of the eyes may occur during a normal blink. The "rider artifact" at the onset of upward saccade is caused by the eyelid as it lags behind the eyes at the beginning of the movement. Smaller rider artifacts, caused by the horizontal asymmetry of the eyelid, can be noted during horizontal but not downward saccades. Techniques that use scaled EOG to remove ocular artifacts from EEG recordings may remove some of the frontal EEG together with the ocular artifacts. Dipole source techniques allow the ocular generators to be distinguished from the nearby brain generators. A problem with dipole source techniques is that the head model used in the calculation is not accurate at the eyes. A new technique uses principal component analysis to estimate the ocular artifact at each electrode without using a head model. This technique is the most effective way to remove ocular artifacts from EEG recordings. (Abstract shortened by UMI.)

The aim of this dissertation was to provide realistic descriptions of two unique properties of the mammalian coronary microcirculation using our novel application of colored microspheres in myocardium. One important objective was to provide an innovative approach to investigate the controversial issue of whether individual coronary arteries communicate at the microvascular level, thus potentially giving rise to a zone of dual arterial supply. Simultaneous in vivo infusion of two distinctly colored microsphere suspensions into the left anterior descending (LAD; red spheres) and left circumflex (LCx; blue spheres) arteries identified a specific interface region of canine myocardium perfused by both arterial branches. Two distinct zones were delineated, and their widths measured. One region was defined as the Interface Transition Zone (ITZ). This region was formed by microvessels supplied by the individual parent arteries, and separated tissue containing only one or the other colored microspheres. The second region defined as the Boundary Watershed Zone (BWZ) was formed by capillaries containing sphere aggregates of both colors; it was located exclusively within the ITZ. In addition, the ITZ and BWZ were significantly wider in subepicardial than in subendocardial regions. Our subsequent study investigated the potential lability of the coronary watershed zones. As before, two differently colored microsphere suspensions were infused into the LAD (red spheres) and LCx (blue spheres) arteries of nine dogs. Subsequently, the LAD was ligated and a third set (green) of spheres was infused into the LCx. Capillaries perfused exclusively by the LAD before occlusion adjacent to the interface contained green microspheres as well as red/green aggregates, indicating lateral extension of the LCx perfusion territory. Results showed that occlusion caused a 24% expansion of the ITZ and a 48% expansion of the BWZ. In addition, all expansions were significantly greater in subepicardial compared to subendocardial regions. These results demonstrated the capability of microvascular anastomoses in providing blood flow to the periphery of an ischemic region. We also applied our colored microsphere technique for the characterization of coronary capillary flow direction in rat myocardium. Our initial study determined capillary flow direction in two groups of male Sprague-Dawley rats: (1) pressure overload hypertrophy and (2) sham-operated controls. Chronic pressure overload was induced in neonatal rats by aortic constriction. Six weeks postsurgery, both groups received sequential in vivo infusion of two differently colored microsphere suspensions into the left atrial appendage. Histological analysis of 40 $\mu$m serial sections revealed that certain coronary capillaries contained microspheres of both colors. A capillary flow vector was established based on the sequence of colors trapped within each aggregate. Examination of flow vectors among neighboring capillaries enabled the characterization of regional capillary flow direction. Results indicated a predominance in concurrent flow, which decreased significantly over a range of capillary rankings. The percentage of concurrent flow was also significantly lower in subendocardium compared with midmyocardium. This study provided previously unattainable data regarding transmural capillary flow direction and suggested regional adaptations in coronary microvascular flow. (Abstract shortened by UMI.)

No description available.

No description available.