Vasoactive Intestinal Peptide Stimulates Catecholamine Biosynthesis in Isolated Adrenal Chromaffin Cells: Evidence for a Cyclic AMP-Dependent Phosphorylation and Activation of Tyrosine Hydroxylase

Vasoactive intestinal peptide (VIP) increased catecholamine biosynthesis in bovine adrenal chromaffin cells by 50–200%. Six related peptides produced no effects. In addition, VIP increased tyrosine hydroxylase (TH) activity measured in gel-filtered supernatants prepared from homogenates of treated cells. The hypothesis that cyclic AMP is the second messenger involved in these effects of VIP was also evaluated. VIP led to an elevation of cyclic AMP levels, and this increase occurred over a similar concentration range and time course as the activation of TH and the increase in catecholamine biosynthesis. Each measure reached maximal levels at 10–20 γM VIP within 1 min and remained elevated for at least 16 min. These changes produced by VIP were paralleled by enhanced phosphorylation of TH, and this phosphorylation occurred on a single tryptic peptide that was the same peptide whose phosphorylation has been previously shown to be stimulated by forskolin. In contrast to VIP and forskolin, 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester known to activate protein kinase C, increased the phosphorylation on a total of three tryptic peptides of TH. Our results indicate that VIP stimulates catecholamine biosynthesis in chromaffin cells through the phosphorylation and activation of TH and support the conclusion that a cyclic AMP-dependent phosphorylation of TH is responsible for these effects.     

In situ phosphorylation of tyrosine hydroxylase in chromaffin cells: Localization to soluble compartments

The present studies investigated the subcellular distribution of acetylcholine's effects upon the phosphorylation of tyrosine hydroxylase in isolated purified bovine adrenal chromaffin cells. After labeling the intact chromaffin cells with ³²P_i, over 90% of the [³²P]tyrosine hydroxylase was found in soluble fractions. Stimulation of the cells with acetylcholine, the natural secretagogue of chromaffin cells, increased the phosphorylation of tyrosine hydroxylase and over 90% of the increase was associated with soluble tyrosine hydroxylase. Homogenates and subcellular fractions from chromaffin cells were also prepared and phosphorylated in vitro in an attempt to optimize detection of tyrosine hydroxylase phosphorylation. In chromaffin cell homogenates, both 8-bromo-cyclic AMP and calcium increased ³²P incorporation into tyrosine hydroxylase, and again over 90% of the increase was observed in soluble fractions. In the particulate fraction, phosphorylation of a band which comigrated with tyrosine hydroxylase in electrophoresis was occasionally detected but only with very long autoradiographic exposures.Tyrosine hydroxylase enzymatic activity in the isolated purified chromaffin cells was also found to be associated predominantly (approx 90%) with soluble fractions. In contrast, a large portion (40–50%) of the tyrosine hydroxylase activity from crude bovine adrenal medullae was associated with the particulate fraction.The data indicate that although tyrosine hydroxylase (and possibly kinases) can associate with particulate fractions when isolated from crude bovine adrenal medullae, the enzyme is predominantly soluble when isolated from the isolated cells. Further, the effects of acetylcholine on the isolated chromaffin cells are predominantly associated with this soluble tyrosine hydroxylase and its attendant kinases.     

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.     

Enhancement of tyrosine hydroxylase phosphorylation and activity by glial cell line-derived neurotrophic factor

Although glial cell-line derived neurotrophic factor (GDNF) acts as a potent survival factor for dopaminergic neurons, it is not known whether GDNF can directly alter dopamine synthesis. Tyrosine hydroxylase (TH) is the rate-limiting enzyme for dopamine biosynthesis, and its activity is regulated by phosphorylation on three seryl residues: Ser-19, Ser-31, and Ser-40. Using a TH-expressing human neuroblastoma cell line and rat primary mesencephalic neuron cultures, the present study examined whether GDNF alters the phosphorylation of TH and whether these changes are accompanied by increased enzymatic activity. Exposure to GDNF did not alter the TH protein level in either neuroblastoma cells or in primary neurons. However, significant increases in the phosphorylation of Ser-31 and Ser-40 were detected within minutes of GDNF application in both cell types. Enhanced Ser-31 and Ser-40 phosphorylation was associated with increased TH activity but not dopamine synthesis in neuroblastoma cells, possibly because of the absence of l-aromatic amino acid decarboxylase activity in these cells. In contrast, increased phosphorylation of Ser-31 and Ser-40 was found to enhance dopamine synthesis in primary neurons. Pharmacological experiments show that Erk and protein kinase A phosphorylate Ser-31 and Ser-40, respectively, and that their inhibition blocked both TH phosphorylation and activity. Our results indicate that, in addition to its role as a survival factor for dopaminergic neurons, GDNF can directly increase dopamine synthesis.     

The protective effect of vitamin D against carbon tetrachloride damage to the rat liver

We investigated the protective effect of vitamin D against liver damage caused by carbon tetrachloride (CCl₄). Twenty-four male rats were divided into four equal groups: G1, untreated controls; G2, administered CCl₄; G3, administered both CCl₄ and vitamin D for 10 weeks; G4, administered CCl₄ for 10 weeks and vitamin D for 12 weeks. At the end of experiment, intracardiac blood samples were taken and liver samples were removed. Hepatic damage due to CCl₄ was assessed using biochemistry and histopathology. Glutathione (GSH) levels decreased, while malondialdehyde (MDA) levels increased in liver tissues of G2. Alanine transaminase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl-transaminase (GGT) levels increased, while albumin (ALB) levels decreased. Hepatocyte degeneration, lobular disorder, sinusoid dilation, focal necrotic areas, hyperemia, and glycogen loss were observed. Hepatic fibrosis was observed around portal areas and central veins. Bridging fibrous septa were formed between portal veins. By immunohistochemistry, both matrix metalloproteinase-9 (MMP-9) and desmin reactivity were increased. All aspects of liver damage were at least partially prevented in rats treated with vitamin D. Vitamin D appears to act as an antioxidant and anti-fibrotic to protect the rat liver against damage.     

Critical thresholds for eventual extinction in randomly disturbed population growth models

This paper considers several single species growth models featuring a carrying capacity, which are subject to random disturbances that lead to instantaneous population reduction at the disturbance times. This is motivated in part by growing concerns about the impacts of climate change. Our main goal is to understand whether or not the species can persist in the long run. We consider the discrete-time stochastic process obtained by sampling the system immediately after the disturbances, and find various thresholds for several modes of convergence of this discrete process, including thresholds for the absence or existence of a positively supported invariant distribution. These thresholds are given explicitly in terms of the intensity and frequency of the disturbances on the one hand, and the population’s growth characteristics on the other. We also perform a similar threshold analysis for the original continuous-time stochastic process, and obtain a formula that allows us to express the invariant distribution for this continuous-time process in terms of the invariant distribution of the discrete-time process, and vice versa. Examples illustrate that these distributions can differ, and this sends a cautionary message to practitioners who wish to parameterize these and related models using field data. Our analysis relies heavily on a particular feature shared by all the deterministic growth models considered here, namely that their solutions exhibit an exponentially weighted averaging property between a function of the initial condition, and the same function applied to the carrying capacity. This property is due to the fact that these systems can be transformed into affine systems.

     

Impact of heterozygote CFTR Mutations in COPD patients with Chronic Bronchitis

Background

Cigarette smoking causes Chronic Obstructive Pulmonary Disease (COPD), the 3rd leading cause of death in the U.S. CFTR ion transport dysfunction has been implicated in COPD pathogenesis, and is associated with chronic bronchitis. However, susceptibility to smoke induced lung injury is variable and the underlying genetic contributors remain unclear. We hypothesized that presence of CFTR mutation heterozygosity may alter susceptibility to cigarette smoke induced CFTR dysfunction. Consequently, COPD patients with chronic bronchitis may have a higher rate of CFTR mutations compared to the general population.

Methods

Primary human bronchial epithelial cells derived from F508del CFTR heterozygotes and mice with (CFTR+/-) and without (CFTR+/+) CFTR heterozygosity were exposed to whole cigarette smoke (WCS); CFTR-dependent ion transport was assessed by Ussing chamber electrophysiology and nasal potential difference measurements, respectively. Caucasians with COPD and chronic bronchitis, age 40 to 80 with FEV₁/FVC < 0.70 and FEV₁ < 60% predicted, were selected for genetic analysis from participants in the NIH COPD Clinical Research Network’s Azithromycin for Prevention of Exacerbations of COPD in comparison to 32,900 Caucasian women who underwent prenatal genetic testing. Genetic analysis involved an allele-specific genotyping of 89 CFTR mutations.

Results

Exposure to WCS caused a pronounced reduction in CFTR activity in both CFTR (+/+) cells and F508del CFTR (+/-) cells; however, neither the degree of decrement (44.7% wild-type vs. 53.5% F508del heterozygous, P = NS) nor the residual CFTR activity were altered by CFTR heterozygosity. Similarly, WCS caused a marked reduction in CFTR activity measured by NPD in both wild type and CFTR heterozygous mice, but the severity of decrement (91.1% wild type vs. 47.7% CF heterozygous, P = NS) and the residual activity were not significantly affected by CFTR genetic status. Five of 127 (3.9%) COPD patients with chronic bronchitis were heterozygous for CFTR mutations which was not significantly different from controls (4.5%) (P = NS).

Conclusions

The magnitude of WCS induced reductions in CFTR activity was not affected by the presence of CFTR mutation heterozygosity. CFTR mutations do not increase the risk of COPD with chronic bronchitis. CFTR dysfunction due to smoking is primarily an acquired phenomenon and is not affected by the presence of congenital CFTR mutations.     

The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues

Proapoptotic Bcl-2 family members have been proposed to play a central role in regulating apoptosis. However, mice lacking bax display limited phenotypic abnormalities. As presented here, bak(-/-) mice were found to be developmentally normal and reproductively fit and failed to develop any age-related disorders. However, when Bak-deficient mice were mated to Bax-deficient mice to create mice lacking both genes, the majority of bax(-/-)bak(-/-) animals died perinatally with fewer than 10% surviving into adulthood. bax(-/-)bak(-/-) mice displayed multiple developmental defects, including persistence of interdigital webs, an imperforate vaginal canal, and accumulation of excess cells within both the central nervous and hematopoietic systems. Thus, Bax and Bak have overlapping roles in the regulation of apoptosis during mammalian development and tissue homeostasis.     

The phylogenetic status of arthropods, as inferred from 18S rRNA sequences

Partial 18S rRNA sequences of five chelicerate arthropods plus a crustacean, myriapod, insect, chordate, echinoderm, annelid, and platyhelminth were compared. The sequence data were used to infer phylogeny by using a maximum-parsimony method, an evolutionary-distance method, and the evolutionary-parsimony method. The phylogenetic inferences generated by maximum-parsimony and distance methods support both monophyly of the Arthropoda and monophyly of the Chelicerata within the Arthropoda. These results are congruent with phylogenies based on rigorous cladistic analyses of morphological characters. Results support the inclusion of the Arthropoda within a spiralian or protostome coelomate clade that is the sister group of a deuterostome clade, refuting the hypothesis that the arthropods represent the "primitive" sister group of a protostome coelomate clade. Bootstrap analyses and consideration of all trees within 1% of the length of the most parsimonious tree suggest that relationships between the nonchelicerate arthropods and relationships within the chelicerate clade cannot be reliably inferred with the partial 18S rRNA sequence data. With the evolutionary-parsimony method, support for monophyly of the Arthropoda is found in the majority of the combinations analyzed if the coelomates are used as "outgroups." Monophyly of the Chelicerata is supported in most combinations assessed. Our analyses also indicate that the evolutionary-parsimony method, like distance and parsimony, may be biased by taxa with long branches. We suggest that a previous study's inference of the Arthropoda as paraphyletic may be the result of (a) having two few arthropod taxa available for analysis and (b) including long-branched taxa.