Alpha 1 adrenergic receptor control of renal blood vessels during aging

Aging humans and rats have a reduced renal vascular constriction response to stress, change in posture, or exercise. In this study, renal interlobar arteries from 9- (intermediate age) to 15-month-old (aging) male Wistar rats constricted less to alpha-adrenergic agonists than those of 4-month-old (young adult) rats. The reduced contraction to A61603 (alpha 1 A agonist) was similar to that to norepinephrine and phenylephrine. Therefore, it appears that the reduction in constriction is primarily related to alpha 1 A receptor stimulation. GeneChip microarray hybridization analysis of the interlobar arteries with the RAE 230A GeneChip indicated that there were no significant differences in gene expression for alpha 1 A/C, 1B, or 1D receptors between 4-month-old (young adult) and 1-year-old (aging) male Wistar rats. Competitive binding experiments (prazosin) revealed that maximal binding (Bmax, fmol/mg protein) of the alpha 1 receptors of interlobar arteries was reduced 25% by 10 months of age and 50% by 18+ months of age. Alpha 1 receptor-induced arterial constriction and prazosin binding were both down-regulated. The loss of receptor-initiated constriction likely includes down-regulation of maximum agonist binding by alpha 1 adrenergic receptors.     

Long-term persistence of male copulatory behavior in castrated and photo-inhibited Siberian hamsters

Gonadal steroids are essential for the long-term maintenance of the full repertoire of sexual behavior in male rodents. Typically, all individuals of several species cease to display the ejaculatory reflex within a few weeks of castration. The present study documents the persistence of the ejaculatory reflex 19 weeks after orchidectomy in 40% of male Siberian hamsters maintained in long or short day lengths; testosterone was undetectable in the circulation of these animals. Intact hamsters transferred from a long to a short photoperiod underwent gonadal regression: 50% of these animals continued to display mating behavior culminating in ejaculation throughout 25 weeks of testing. The remaining animals failed to ejaculate after approximately 11 weeks of short day treatment but resumed mating coincident with spontaneous gonadal recrudescence. Activation of sex behavior in the latter cohort appears to depend on gonadal steroids and is in contrast to the copulatory behavior of the substantial proportion of the study population that sustains the full sexual repertoire in the long-term absence of gonadal steroids. Sex behavior of the latter animals may be dependent on nongonadal steroids or mediation by steroid-independent mechanisms.     

Thromboxane A2 induces airway constriction through an M3 muscarinic acetylcholine receptor-dependent mechanism

Thromboxane A2 (TXA2) is a potent lipid mediator released by platelets and inflammatory cells and is capable of inducing vasoconstriction and bronchoconstriction. In the airways, it has been postulated that TXA2 causes airway constriction by direct activation of thromboxane prostanoid (TP) receptors on airway smooth muscle cells. Here we demonstrate that although TXA2 can mediate a dramatic increase in airway smooth muscle constriction and lung resistance, this response is largely dependent on vagal innervation of the airways and is highly sensitive to muscarinic acetylcholine receptor (mAChR) antagonists. Further analyses employing pharmacological and genetic strategies demonstrate that TP-dependent changes in lung resistance and airway smooth muscle tension require expression of the M2 mAChR subtype. These results raise the possibility that some of the beneficial actions of anticholinergic agents used in the treatment of asthma and chronic obstructive pulmonary disease result from limiting physiological changes mediated through the TP receptor. Furthermore, these findings demonstrate a unique pathway for TP regulation of homeostatic mechanisms in the airway and suggest a paradigm for the role of TXA2 in other organ systems.     

Expression and function of NPSR1/GPRA in the lung before and after induction of asthma-like disease

A genetic contribution to asthma susceptibility is well recognized, and linkage studies have identified a large number of genes associated with asthma pathogenesis. Recently, a locus encoding a seven-transmembrane protein was shown to be associated with asthma in founder populations. The expression of the protein GPRA (G protein-coupled receptor for asthma susceptibility) in human airway epithelia and smooth muscle, and its increased expression in a mouse model of asthma, suggested that a gain-of-function mutation in this gene increased the disease risk. However, we report here that the development of allergic lung disease in GPRA-deficient mice is unaltered. A possible explanation for this finding became apparent upon reexamination of the expression of this gene. In contrast to initial studies, our analyses failed to detect expression of GPRA in human lung tissue or in mice with allergic lung disease. We identify a single parameter that distinguishes GPRA-deficient and wild-type mice. Whereas the change in airway resistance in response to methacholine was identical in control and GPRA-deficient mice, the mutant animals showed an attenuated response to thromboxane, a cholinergic receptor-dependent bronchoconstricting agent. Together, our studies fail to support a direct contribution of GPRA to asthma pathogenesis. However, our data suggest that GPRA may contribute to the asthmatic phenotype by altering the activity of other pathways, such as neurally mediated mechanisms, that contribute to disease. This interpretation is supported by high levels of GPRA expression in the brain and its recent identification as the neuropeptide S receptor.     

Synthesis of acid-cleavable light isotope-coded affinity tags (ICAT-L) for potential use in proteomic expression profiling analysis

A convenient synthesis of some homologous light isotope-coded affinity tags (ICAT-L) containing an acid-labile moiety between the affinity component biotin and an electrophilic polar linker is described. These light ICAT reagents give smooth mass spectral signals in tandem mass spectrometry (MS/MS) analyses of some commercially available cysteine-containing peptides. However, these ICAT molecules are designed for use in identification and relative quantification of whole or partially purified cellular and tissue proteomes. Since the biotin moiety can be readily cleaved off the reagent after mass tagging, undesired residual fragmentation patterns caused by biotin of derived peptides, as normally observed using biotin-containing ICAT reagents, are effectively eliminated. This strategy should enhance peptide sequence coverage significantly which, in turn, should result in improving the quality of data obtained during data-dependent peptide mass and tandem mass spectral analysis of whole proteomes.     

Deletion of ripA alleviates suppression of the inflammasome and MAPK by Francisella tularensis

Francisella tularensis is a facultative intracellular pathogen and potential biothreat agent. Evasion of the immune response contributes to the extraordinary virulence of this organism although the mechanism is unclear. Whereas wild-type strains induced low levels of cytokines, an F. tularensis ripA deletion mutant (LVSΔripA) provoked significant release of IL-1β, IL-18, and TNF-α by resting macrophages. IL-1β and IL-18 secretion was dependent on inflammasome components pyrin-caspase recruitment domain/apoptotic speck-containing protein with a caspase recruitment domain and caspase-1, and the TLR/IL-1R signaling molecule MyD88 was required for inflammatory cytokine synthesis. Complementation of LVSΔripA with a plasmid encoding ripA restored immune evasion. Similar findings were observed in a human monocytic line. The presence of ripA nearly eliminated activation of MAPKs including ERK1/2, JNK, and p38, and pharmacologic inhibitors of these three MAPKs reduced cytokine induction by LVSΔripA. Animals infected with LVSΔripA mounted a stronger IL-1β and TNF-α response than that of mice infected with wild-type live vaccine strain. This analysis revealed novel immune evasive mechanisms of F. tularensis.     

Pioglitazone prevents cardiac remodeling in high-fat, high-calorie-induced Type 2 diabetes mellitus

The agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma) ameliorate cardiovascular complications associated with diabetes mellitus. We tested the hypothesis that recovery from ailing to failing myocardium in diabetes by PPARgamma agonist is in part due to decreased matrix metalloproteinase-9 (MMP-9) activation and left ventricular (LV) tissue levels of homocysteine (Hcy). C57BL/6J mice were made diabetic (D) by feeding them a high-fat calorie diet. PPARgamma was activated by adding pioglitazone (Pi) to the diet. After 6 wk, mice were grouped into: normal calorie diet (N), D, N + Pi and D + Pi (n = 6 in each group). LV variables were measured by echocardiography, endothelial-myocyte (E-M) coupling was measured in cardiac rings, and MMP-9 activation was measured by zymography. Blood glucose levels were twofold higher in D mice compared with N mice. Pi decreased the levels of glucose in D mice to the levels in N mice. LV Hcy levels were 3.5 +/- 0.5 microM in N groups compared with 12.4 +/- 0.6 microM in D groups. Treatment with Pi normalized the LV levels of Hcy but had no effect on plasma levels of Hcy. In the D group, LV contraction was reduced compared with that of the N group and was ameliorated by treatment with Pi. LV wall thickness was reduced to 0.25 +/- 0.02 mm in the D group compared with 0.42 +/- 0.01 mm in the N group. LV diastolic diameter was 3.05 +/- 0.01 mm in the D group compared with 2.20 +/- 0.02 mm in the N group. LV systolic diameter was 1.19 +/- 0.02 mm in the D group and 0.59 +/- 0.01 mm in the N group. Pi normalized the LV variables in D mice. The responses to ACh and nitroprusside were attenuated in diabetic hearts, suggesting that there was E-M uncoupling in the D group compared with the N group, which was ameliorated by Pi. Plasma and LV levels of MMP-2 and -9 activities were higher in the D group than in the N group but normalized after Pi treatment. These results suggest that E-M uncoupling in the myocardium, in part, is due to increased MMP activities secondary to suppressing PPARgamma activity in high-fat, calorie-induced Type 2 diabetes mellitus.     

Distribution of current photosynthate in two Guinea grass (Panicum maximum Jacq.) cultivars

In a glasshouse experiment, different tiller categories (main, young primary, and old primary) of two Guinea grass cultivars, Momba?a and Tanzania, were (14)C-labelled to investigate C translocation between tillers. In both cultivars, young primary tillers retained less radiocarbon (79%) than main (86%) and old primary (87%) labelled tillers, suggesting that the photosynthetic capacity of the young tillers exceeds their capacity either to store or use that photosynthate for growth and maintenance. In cv. Tanzania the old primary tillers translocated more photoassimilate to their daughter tillers and to the rest of the plant than cv. Momba?a, suggesting either higher vascular connectivity between tillers or stronger diurnal fluctuations in sink-source balance in Tanzania than Momba?a. For unlabelled tillers, specific activities were almost always higher in roots than in shoots, although total radiocarbon uptake by roots was reduced in younger tillers with low root mass. Where young primary tillers were labelled, the largest single repository of exported photoassimilate was the main tiller roots, indicating that daughter tillers may make an important contribution to the maintenance of older roots on mature tillers.