We evaluated the activity of tissue (i.e., brain and cardiac) versus circulatory renin angiotensin systems (RASs) during (1) the development of high salt-induced hypertension and cardiac hypertrophy; and (2) the prevention of salt-sensitive hypertension by chronic blockade of brain "ouabain". For protocol I, ACE mRNA and activity in the hypothalamus and pons, as well as in the left ventricle (LV) and right ventricle (RV); angiotensin I and II (Ang I, II) in the hypothalamus and pons, as well as in the ventricle and plasma, and plasma renin activity (PRA) were assessed in Dahl salt-sensitive (Dahl S) and salt-resistant (Dahl R) rats on high (1370 mumol/g) or regular salt (120 mumol/g) diet for 2 or 4--5 weeks. For protocol II, ACE mRNA and activity in the hypothalamus and pons, and in the LV and RV were measured in Dahl S rats on high salt with either icv Fab fragments binding brain "ouabain" or gamma-globulins as control, or, regular salt with icv gamma-globulins; and in Dahl R rats on high or regular salt diet for 4--5 weeks. (Abstract shortened by UMI.)

Cytosolic calcium (Ca2+) levels are critical for the control of muscle contraction and are tightly regulated by a variety of Ca 2+ transport systems localized in various membranes. Ca2+ binding proteins such as calmodulin (CaM) and Ca2+/CaM-dependent protein kinases (CaM Kinases) are believed to exert major regulatory control on Ca2+ activity. Previous studies in this lab led to the cloning of a cDNA encoding a CaM Kinase II beta isoform from skeletal muscle that differed from the classical beta isoform by the inclusion of three alternatively spliced exons in the variable domain which were enriched in proline residues. A CaM Kinase, assumed to be localized in the sarcoplasmic reticulum (SR), has been implicated in the regulation of excitation-contraction (E-C) coupling. We hypothesized that this novel CaM Kinase II beta isoform called SOCK (Son Of CaM Kinase) may be the CaM Kinase II isoform that regulates E-C coupling by being targeted to specific regions of the SR, whereby it phosphorylates critical Ca2+ transporting proteins such as the ryanodine (RyR) and dihydropyridine (DHPR) receptors in response to changes in Ca2+ levels. (Abstract shortened by UMI.)

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder which results in the selective loss of the medium spiny neurons in the striatum. The neuropathological and behavioural deficits of HD can be modeled in rats by injection of the NMDA receptor agonist quinolinic acid (2,3 pyridine decarboxylate; QA) directly into the striatum. The first aim of this study was to examine whether intrastriatal QA injections result in neurons dying by apoptosis as assessed by caspase activation. Animals were intrastriatally injected with QA and striatal brain sections processed for several apoptotic markers. Recently, a novel family of inhibitor of apoptosis (IAP) proteins have been identified and subsequently shown to attenuate cell death induced by a variety of apoptotic triggers in vitro. The goal of the second study was to determine whether adenovirally-mediated overexpression of the IAPs resulted in protection against QA-induced degeneration. The goal of the third study was to determine whether neurons saved by overexpression of the IAPs were still functional. In a third study, animals were intrastriatally injected with recombinant adenoviral constructs containing either xiap, naip, or lacZ one week prior to QA injections (120 nmol). (Abstract shortened by UMI.)

In chromaffin cells, cortical F-actin disassembly in response to stimulation allows the movement of secretory vesicles towards exocytotic sites. Scinderin (Sc), a Ca2+-dependent F-actin severing protein, controls F-actin dynamics. The Sc gene has previously been cloned in our laboratory. Sc has six domains with three actin-binding sites in domains 1, 2 and 5, two PIP2 in domains 1 and 2 and two Ca2+-binding sites. In order to obtain additional data on the role of Sc domains in secretion, we performed experiments with a human growth hormone reporter gene (hGH) system for regulated exocytosis, and different GFP-fusion Sc constructs: Sc1--6, Sc1--2 (the first two domains of Sc), Sc5 (5th domain of Sc), ScL5 (5th domain lacking the third actin binding site of Sc), and ScABS3. Cortical F-actin distribution evaluated by rhodamine-phalloidine staining, in resting and stimulated cells (56 mM K+), showed that cells overexpressing Sc1--6 or Sc1--2 had an increased F-actin disassembly upon stimulation, whereas cells overexpressing Sc5 or ScABS3 had a decreased F-actin disassembly. No significant difference in F-actin assembly/disassembly was obtained for cells overexpressing ScL5. Increased hGH release in response to stimulus was found for cells overexpressing Sc1--6 or Sc1--2 when compared to cells transfected with vector alone. Cells overexpressing Sc5 or ScABS3 showed decreased hGH release in response to stimulus. These results suggest that during secretion the N-terminal half of the protein involved in F-actin severing and the C-terminal domains of Sc are responsible for actin polymerization. Thus, it appears that Sc functions as a molecular switch in the control of actin dynamics during secretion.

TaxolRTM 1a, a very important drug used in cancer chemotherapy, has been synthesized by several groups. However these syntheses are long and there is still the need for a short and efficient route that would allow the production of this compound in larger amount. The route proposed by our group toward a simplified model compound 78 implied a double Diels-Alder strategy and started from readily available protected (L)-arabinose 118 or (D)-guluno-gamma-lactone 146. Conversion of 118 to 164, followed by Diels-Alder reaction led to the stereoselective construction of 165 . Deprotection of the isopropylidene acetal tether group and oxidative cleavage gave compounds 172a and 173a. We were able to ultimately get to compound 189. Despite precedents in the literature the second Diels-Alder cycloaddition required for the construction of rings A and B failed.

$\alpha\beta$ and $\gamma\delta$ T cells are both postulated to play a role in the pathogenesis of Multiple Sclerosis (MS). IFN-$\beta$-1b (Betaseron), a novel treatment for MS, can change the natural course of MS disease, though its exact mechanism of action is unknown. Since any benefit probably involves effects of IFN-$\beta$-1b on $\alpha\beta$ or $\gamma\delta$ T cells, I derived T cells from MS patients and healthy controls and investigated the effects of IFN-$\beta$-1b on their activation, proliferation and activation-induced apoptosis. PHA, anti-CD3 and tetanus toxoid were used as stimulants for one or both T cell types in the presence of 0-1000 IU/mL IFN-$\beta$-1b. Results indicate that IFN-$\beta$-1b differentially affects $\alpha\beta$ and $\gamma\delta$ T cells both in terms of activation and apoptosis, and that MS cells exhibit differential sensitivity to these effects compared to controls. (Abstract shortened by UMI.)

Retinoids have been shown to inhibit the growth of estrogen receptor-positive human breast cancer cells. In this study, it is shown that all-trans-retinoic acid (RA) inhibits the growth of MCF-7 cells by 60%. In these cells, we also found that RA inhibited estrogen-induced transcription. In transient transfection experiments using the vitellogenin-estrogen response element-CAT reporter construct, RA inhibited estrogen-induced transcription by 55%. Cotransfection of wildtype RAR$\alpha$ resulted in a 75% inhibition of estrogen-induced transcription. In order to investigate the mechanisms involved in RA-mediated inhibition of transcription, deletion mutants of RAR$\alpha$ were constructed. Point mutants of RAR$\alpha$ were also evaluated to determine their effects on RA-mediated inhibition of estrogen-induced transcription. Cotransfection of RAR$\alpha$ deletion mutants terminating after amino acid 414 resulted in wildtype inhibition. Using mutants terminating before amino acid 412, the inhibition was significantly reduced. RAR$\alpha$ mutants lacking amino acids 413 and 414 also showed significantly decreased inhibition. The amino acids 413 and 414 correspond to the activating function-2 (AF-2) region in the C-terminus of the RAR$\alpha.$ These results suggest that the RA-mediated inhibition of estrogen-induced transcription in human breast carcinoma cells is mediated by the AF-2 region of the RAR$\alpha.$ MCF-7 cells, stably transfected with a dominant negative RAR$\alpha,$ are growth inhibited by only 25% compared to 60% for untransfected or mock-transfected cells. Taken together, these results suggest that RA-mediated inhibition of estrogen-induced transcription is mediated by the AF-2 region of the RAR$\alpha$ and may play a role in RA-induced inhibition of estrogen receptor-positive human breast cancer cell growth.

Nicotinic stimulation of chromaffin cells causes disassembly of cortical F-actin networks and redistribution of scinderin, a Ca$\sp{2+}$-dependent F-actin severing protein. These Ca$\sp{2+}$-dependent events precede exocytosis. Activation of scinderin by Ca$\sp+$ may cause severing of F-actin and disassembly of actin networks leaving cortical areas of low cytoplasmic viscosity which are the sites of exocytosis. Histamine is a known chromaffin cell secretagogue which induces Ca$\sp+$-dependent release of catecholamines. However, conflicting evidence exists as to the source of Ca$\sp{2+}$ utilized in histamine-evoked secretion. Here we report that histamine-H$\sb1$ receptor activation induces scinderin redistribution, F-actin disassembly and catecholamine release. Histamine evoked similar patterns of distribution of scinderin and filamentous actin. The rapid responses to histamine occurred in the absence of extracellular Ca$\sp{2+}$ and were triggered by release of Ca$\sp{2+}$ from intracellular stores. The trigger for the release of Ca$\sp{2+}$ was inositol 1,4,5-triphosphate (IP$\sb3$) since U-73122, a phospholipase C inhibitor, but not its inactive isomer (U-73343), inhibited the increases in IP$\sb3$, intracellular Ca$\sp{2+}$, scinderin redistribution, cortical F-actin disassembly and catecholamine release in response to histamine. Thapsigargin, an agent known to mobilize intracellular Ca$\sp{2+}$, blocked the rise in intracellular Ca$\sp{2+}$, scinderin redistribution, F-actin disassembly and catecholamine secretion in response to histamine. However, thapsigargin did not modify the increase in IP$\sb3$ induced by histamine. Calphostin C and chelerythrine, two inhibitors of protein kinase C, blocked all responses to histamine with the exception of the release of Ca$\sp{2+}$ from intracellular stores. This suggests that protein kinase C is involved in histamine induced response. The results also show that without F-actin disassembly, raises in intracellular Ca$\sp{2+}$ are not by themselves capable of triggering catecholamine release.

Hypothesis. High sodium diet in rats may induce intermittent sympathetic activation through increasing nighttime water intake, and may enhance cardiac adrenergic responses via an increase in adrenoceptor activity, which may contribute to the development of high sodium diet-induced left ventricular hypertrophy (LVH). Method. In conscious WKY rats, resting hemodynamics and after blocking baroreflex by hexamethonium (Hex), hemodynamic responses to $\alpha\sb1$-, or $\beta$-agonist (phenylephrine, Phe, or isoproterenol, Iso), and then ventricular weights and LV dimensions were determined after 1, 2, 4 or 6 wk medium high or high sodium diet (2 or 8% NaCl) with or without ig administration of $\alpha\sb1$- or $\beta$-blockers (60 $mg/kg/day$ terazosin, Tz, 25 or 100 $mg/kg/day$ nadolol, Nd, alone or both Tz and Nd). Discussion. High sodium diet for 2 to 6 wk induces concentric LVH, without changing most resting hemodynamics including LV filling pressures. Vasodilatation by $\beta$-agonist was attenuated and negative chronotropy of $\alpha\sb1$-agonist augmented by a longer salt exposure, which may indicate a decreased vascular $\beta\sb2$- or an increased sinoatrial node $\alpha\sb1$-receptor responses. Effects of ganglionic and adrenergic blockades suggest a salt-augmented sympathetic control on peripheral arterial resistance. Chronic $\alpha\sb1$- and/or $\beta$-blockers failed to prevent the high sodium diet-induced LVH, which suggests that adrenoceptor activation does not play a primary role in evoking this LVH. However, the blockers shifted the LVH from a concentric to an eccentric form, and Tz augmented the salt-increased water intake. It is plausible that a volume overload-induced hypertrophy associated with the blockers may have masked a reduction in the high sodium diet-induced LVH. (Abstract shortened by UMI.)

Neuropeptide Y (NPY) is a 36 amino acid peptide that is co-stored and co-released with noradrenaline from perivascular sympathetic nerves. In the present project, I tested the hypothesis that the vasopressor effect of NPY is related to its modulation of ion channel activities. The effects of NPY on Ca$\sp{2+}$-channel currents in freshly isolated vascular smooth muscle cells (VSMC) from the rat tail artery were studied with the perforated-patch recording technique. At a testing potential of +10 mV, the amplitude of inward current was increased by 46% in the presence of NPY 150 nM. The effects of NPY on both Ca$\sp{2+}$ current amplitude and steady-state activation were much weaker compared to the dihydropyridine agonist Bay K 8644. Unlike Bay K 8644 which shifted steady-state inactivation curve toward less positive membrane potential by 14.3 mV, NPY had no signifiant effect on steady-state inactivation of the current. A synergistic action between NPY and Bay K 8644 was observed as the combined effect of NPY and Bay K 8644 was larger than the sum of NPY and Bay K 8644 alone. The effects of NPY on the Ca$\sp{2+}$-activated K$\sp+$ channel (K$\sb{(Ca)}$) in VSMCs from the rat tail artery were also studied by whole-cell and single channel recording technique. NPY did not affect the open times or current amplitude, but increased significantly the short (from 0.49 to 0.58 ms) and long (from 441 to 728 ms) close times. An inhibition of K$\sb{(Ca)}$ channels was also observed in cell-attached patches when NPY was applied in the bath solution, indicating involvement of diffusible second messenger. The effect of NPY on K$\sb{(Ca)}$ channels was ATP-dependent. Tyrosine kinase inhibitors (genistein, lavendustin A, and tyrphostin A25) by themselves potentiated K$\sb{(Ca)}$ channel currents in a dose-dependent manner. At a testing potential of +30 mV and in the presence of genistein 15 $\mu$M, whole-cell current amplitude of K$\sb{(Ca)}$ increased by 143%. Similarly, single channel open probability increased by 120%. The potentiating effect of genistein on K$\sb{(Ca)}$ channels was ATP-dependent. Daidzein, an inactive analogue of genistein had no effect on K$\sb{(Ca)}.$ It is concluded that NPY potentiates vasoconstriction by a synergistic effect on C$\sp{2+}$ and K$\sb{(Ca)}$ channels in VSMCs. NPY can promote Ca$\sp{2+}$ entry by increasing Ca$\sp{2+}$ channel activity. Inhibition of K$\sb{(Ca)}$ channel would further increase the influx of C$\sp{2+}$ by promoting membrane depolarization and/or prolonging the duration of action potentials. It is also concluded that the effect of NPY on K$\sb{(Ca)}$-channel is mediated by a phosphorylation-dependent pathway, probably involving tyrosine phosphorylation. (Abstract shortened by UMI.)