No description available.

This thesis examines in vivo and in vitro the effects of prolonged exposure of target cells to PTH on PTH receptors and postreceptor components of the adenylate cyclase system. Using a vitamin D-deficient ($-$D) rat model, hyperparathyroidism in vivo resulted in a decreased number of PTH receptors in kidney and a decreased amount of G protein $ alpha$ subunits. The decreased amount of G$ sb{ rm s} alpha$ was shown to be specific for PTH target tissue and may have played a role in the heterologous desensitization of CT-stimulated adenylate cyclase, demonstrated in renal membranes from the $-$D rats. The study of the control of PTH receptors was pursued using an osteosarcoma clonal cell line, UMR-106, in vitro. Initial characterization of these cells revealed abundant, saturable, cell surface PTH receptors linked to the adenylate cyclase system. Demonstration that the majority of PTH binding was associated with morphologically distinct cells in the UMR-106 population indicated that PTH receptors may be maximally expressed during specific stages in the cell cycle. PTH receptors in UMR-106 cells were shown to be regulated by distinct homologous and heterologous mechanisms. PTH-mediated, homologous desensitization was associated with down-regulation of PTH receptors and loss of G$ sb{ rm s} alpha$ protein form the cell membrane. Heterologous desensitization of PTH responses by PGE$ sb2$ was shown to be cAMP mediated, resulting in a reversible modification of the PTH receptors. This work has demonstrated that multiple mechanisms exist for the regulation of PTH responses both in vivo and in vitro that involve modifications of both the PTH receptors and postreceptor components of the adenylate cyclase system.

These studies investigated possible relief of spasticity in hemiparetic subjects by transcutaneous electrical nerve stimulation (TENS) and its underlying mechanisms. The first two studies quantified the disorders in reflex and voluntary motor functions and addressed the reproducibility of their measurement and their correlation with spasticity scores. Soleus stretch reflexes were enhanced and isometric voluntary contraction force was decreased linearly with increasing spasticity. The last two studies addressed the effects of single and repetitive TENS stimulation on spasticity, reflex and isometric voluntary contractions. Compared to placebo stimulation, single 45 min sessions of TENS prolonged H and stretch reflex latencies for up to 60 min following stimulation. Repetitive (15 daily, 60 min) applications significantly decreased spasticity scores, Hvib/Hctl ratios, stretch reflexes and co-contraction while improving dorsiflexion force. The improvement in spasticity and voluntary motor control may partly have been mediated by presynaptic inhibition and reduced hyperactive stretch reflexes thereby 'unmasking' descending control.

No description available.

No description available.

The cystic fibrosis transmembrane conductance regulator (CFTR) constitutes an ohmic chloride channel, the gating of which is dependent on cAMP-dependent phosphorylation of a regulatory (R) domain and ATP binding and hydrolysis by two nucleotide binding domains (NBDs). Previous studies have suggested that CFTR activation results from PKA phosphorylation of the R-domain, which either relieves its inhibition of the channel, or induces a positive catalytic effect of this domain on ATP dependent gating. In this study, the function of the R-domain was examined using a CFTR variant lacking the entire R-domain. These split channels assemble from separate polypeptides corresponding to the two halves of CFTR after translation of a single mRNA. Even though the back half of the split CFTR channel is expressed as a core glycosylated protein, both halves are able to fold independently and associate with one another to form a PKA-independent, constitutively active channel; however, this occurs with low efficiency. (Abstract shortened by UMI.)

Follicle stimulating hormone (FSH) acting on Sertoli cells of the testis plays important roles during reproductive development. FSH-R knockout (FORKO) mice provide a model to examine alterations in testicular and epididymal structure and function in its absence. Examination of the FORKO testis revealed a gross alteration of Sertoli cell structure indicative of a fluid imbalance. Functional parameters, such as ABP secretion were also significantly reduced in FORKO testis. Morphometry revealed quantitative reductions in seminiferous tubule size. The epididymal epithelium, appeared abnormal and morphometry revealed that epididymal tubule size was reduced in the knockout. Computer Assisted Sperm Analysis on sperm from the cauda epididymidis revealed significant alterations in parameters corresponding to sperm motility as well as sperm counts. These data suggest an important role for the FSH receptor on Sertoli cell structure and functions and on epididymal epithelial size and functions in relation to sperm motility.

Thalassemia is a blood disorder requiring lifelong transfusions for survival. Erythrocytes accumulate toxic iron at their membranes, triggering an oxidative cascade that leads to their premature destruction. We hypothesized that removing this proximate iron compartment as a primary treatment using novel iron chelators, could prevent hastened red cell removal and clinically alleviate the need for transfusion. Novel, highly cell permeable iron chelators, pyridoxal isonicotinoyl hydrazone (PIH) and pyridoxal ortho-chlorobenzoyl hydrazone (o-108) were compared to the present mainstay, desferrioxamine (DFO) and deferiprone (L1), in vitro and in vivo . Treatment of human model beta-thalassemic erythrocytes with chelators resulted in significant depletion of membrane-associated iron and reduced oxidative stress as indicated by a decrease in methemoglobin levels. When administered to beta-thalassemic mice, iron chelators mobilized erythrocyte membrane iron, reduced cellular oxidation, and prolonged erythrocyte survival. Consistently, these mice showed improved hematological abnormalities. A beneficial effect as early as the erythroid precursor stage was also determined by normalized proportions of mature versus immature reticulocytes. Remarkably, all four chelators reduced iron accumulation in target organs. Most importantly, o-108 revealed superior activity, decreasing iron in liver and spleen by ~5-fold and ~2-fold, respectively, compared to DFO. Our study demonstrates that iron chelators ameliorate thalassemia in a human and murine model, and validates their primary use as an alternative to transfusion therapy.

In glomerular epithelial cells (GEC), the actin cytoskeleton is a key determinant of cell morphology and functions, including permselectivity. Complement C5b-9 induces sublytic GEC injury associated with GEC morphological changes and proteinuria. This study addressed the role of Rho GTPases in complement-mediated GEC injury. We demonstrated that the amount of active RhoA increased; while the amount of active Rac1 and Cdc42 were decreased in C5b-9 mediated sublytic GEC injury both in vitro and in glomeruli from rats with PHN in vivo. Complement mediated inactivation of p190RhoGAP may contribute to complement-induced RhoA activation. Overexpression of constitutively active or dominant negative mutants of RhoA, Rac1 and Cdc42 distinctly altered GEC morphology and F-actin pattern. Complement caused changes in GEC actin cytoskeleton, at least in part mediated by a downstream kinase of RhoA--Rho kinase (ROCK). Activation of RhoA exacerbated complement-mediated cytotoxicity in GEC, while inhibition of ROCK attenuated it.

Airway hyperresponsiveness (AHR) is a key feature of asthma, characterized by exaggerated rate and extent of shortening of airway smooth muscle. Two isoforms of the smooth muscle myosin differ by the presence [(+)insert] or absence [(-)insert] of a 7 amino acid insert. The (+)insert exhibits a 2-fold greater ATPase activity and velocity of actin filament propulsion in the in vitro motility assay. The expression of these isoforms and other contractile proteins was quantified in the trachea of the Fisher and Lewis rat model of innate AHR. We found 95% greater mRNA and 45% greater protein expression of the (+)insert isoform in the trachea of the hyperresponsive Fisher animal (p<0.01), but no difference in other contractile proteins. A greater extent of myosin phosphorylation was also observed (55.1+/- d6.4 vs. 41.4+/-d6.1, p<0.01). These results suggest that in addition to greater myosin activation, an increased expression of the (+)insert isoform contribute to AHR.