Aromatic l‐amino acid decarboxylase phosphorylation and activation by PKGIαin vitro

Soluble guanylyl cyclase (sGC) is the major physiological receptor for nitric oxide (NO) throughout the central nervous system. Three different subunits form the α₁/β₁ and α₂/β₁ heterodimeric enzymes that catalyze the reaction of GTP to the second messenger cGMP. Both forms contain a prosthetic heme group which binds NO and mediates activation by NO. A number of studies have shown that NO/cGMP signaling plays a major role in neuronal cell differentiation during development of the central nervous system. In the present work, we studied regulation and expression of sGC in brain of rats during postnatal development using biochemical methods. We consistently observed a surprising decrease in cerebral NO sensitive enzyme activity in adult animals in spite of stable expression of sGC subunits. Total hemoprotein heme content was decreased in cerebrum of adult animals, likely because of an increase in heme oxygenase activity. But the loss of sGC activity was not simply because of heme loss in intact heterodimeric enzymes. This was shown by enzyme activity determinations with cinaciguat which can be used to test heme occupancy in intact heterodimers. A reduction in heterodimerization in cerebrum of adult animals was demonstrated by co‐precipitation analysis of sGC subunits. This explained the observed decrease in NO sensitive guanylyl cyclase activity in cerebrum of adult animals. We conclude that differing efficiencies in heterodimer formation may be an important reason for the lack of correlation between sGC protein expression and sGC activity that has been described previously. We suggest that heterodimerization of sGC is a regulated process that changes during cerebral postnatal development because of still unknown signaling mechanisms.     

Developmental changes of nitric oxide-sensitive guanylyl cyclase expression in pulmonary arteries

Inhaled nitric oxide (NO) is known to influence the contractile state of pulmonary arteries most likely by activation of soluble guanylyl cyclase (sGC) in smooth muscle cells. However, the cellular distribution of sGC has not been determined empirically, due to a lack of specific antibodies. Here, we describe a novel antibody directed against the beta1 subunit of sGC to study the cellular distribution of sGC in lung during development. Using the novel antibody, the enzyme was demonstrated in fetal, neonatal, and adult lungs by Western blot, showing maximum expression in neonatal lung. These data were confirmed by measurements of sGC activity. In pulmonary arteries of fetal lung sGC-beta1 immunoreactivity was present in smooth muscle cells and absent in endothelial cells. With postnatal development an increase in immunoreactivity in endothelial cells and a reciprocal decrease in smooth muscle cells was apparent. The reported changes in sGC expression likely contribute to the known age-dependent differences in response to inhaled NO.     

Age-specific pulmonary cytochrome P-450 3A1 expression in postnatal and adult rats

A major cause of death and illness in children under the age of five, most living in polluted cities, is respiratory disease. Previous studies have shown that neonatal animals are more susceptible to bioactivated pulmonary cytotoxicants than adults, despite lower expression of the pulmonary cytochrome P-450s (CYP450s) thought to be involved in bioactivation. One CYP450 that is well documented in the bioactivation of many drugs and environmental toxicants in adult lung, but whose expression has not been evaluated during postnatal pulmonary development, is CYP450 3A (CYP3A). We compared age-specific expression of CYP3A1 in 7-day-old and adult male Sprague-Dawley rats. Unlike those shown for previously studied pulmonary CYP450s, expression levels for CYP3A1 mRNA in differentiating airway cells of postnatal rats are the same as in fully differentiated airway cells of adults. CYP3A1 protein expression (28%) and enzymatic activity (23%) were lower in postnatal airways compared with adults. Although other CYP450 immunoreactive proteins are primarily expressed in nonciliated cells, immunoreactive CYP3A1 protein was expressed in both ciliated and nonciliated cells in postnatal and adult rat proximal airways. CYP3A1 protein is detected diffusely throughout ciliated and nonciliated cells in 7-day-old rats, whereas it is only detected in the apex of these cells in adult rats. This study demonstrates that the lungs of postnatal rats have detectable levels of CYP3A1 and that CYP3A1 mRNA expression appears not to be age dependent, whereas steady-state CYP3A1 protein levels and enzyme activity show an age-dependent pattern.     

Dependence of soluble guanylyl cyclase activity on calcium signaling in pituitary cells

The role of nitric oxide (NO) in the stimulation of soluble guanylyl cyclase (sGC) is well established, but the mechanism by which the enzyme is inactivated during the prolonged NO stimulation has not been characterized. In this paper we studied the interactions between NO and intracellular Ca(2+) in the control of sGC in rat anterior pituitary cells. Experiments were done in cultured cells, which expressed neuronal and endothelial NO synthases, and in cells with elevated NO levels induced by the expression of inducible NO synthase and by the addition of several NO donors. Basal sGC-dependent cGMP production was stimulated by the increase in NO levels in a time-dependent manner. In contrast, depolarization of cells by high K(+) and Bay K 8644, an L-type Ca(2+) channel agonist, inhibited sGC activity. Depolarization-induced down-regulation of sGC activity was also observed in cells with inhibited cGMP-dependent phosphodiesterases but not in cells bathed in Ca(2+)-deficient medium. This inhibition was independent from the pattern of Ca(2+) signaling (oscillatory versus nonoscillatory) and NO levels, and was determined by averaged concentration of intracellular Ca(2+). These results indicate that inactivation of sGC by intracellular Ca(2+) serves as a negative feedback to break the stimulatory action of NO on enzyme activity in intact pituitary cells.     

Soluble guanylyl cyclase during postnatal porcine pulmonary maturation

The nitric oxide (NO)/cGMP pathway plays a key role in the regulation of pulmonary vascular tone during the transition from the fetal to the neonatal circulation, and it is impaired in pathophysiological conditions such as pulmonary hypertension. In the present study, we have analyzed the changes in the function and expression of soluble guanylyl cyclase (sGC) in pulmonary arteries during early postnatal maturation in isolated third-branch pulmonary arteries from newborn (3-18 h of age) and 2-wk-old piglets. The expression of sGC beta(1)-subunit in pulmonary arteries increased with postnatal age both at the level of mRNA and protein. The catalytic region of porcine sGC beta(1) was sequenced, showing a 92% homology with the human sequence. This age-dependent increase in sGC expression correlated with increased vasorelaxant responses to the physiological sGC activator NO and to the exogenous sGC activator YC-1, but not to the membrane-permeable cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate. In conclusion, an increased expression of sGC in pulmonary conduit arteries from 2-wk-old compared with newborn piglets explains, at least partly, the age-dependent increase in the vasorelaxant response of NO and other activators of sGC.     

Regulation of cGMP-dependent protein kinase expression by soluble guanylyl cyclase in vascular smooth muscle cells

Vascular smooth muscle cells (VSMC) undergo many phenotypic changes when placed in culture. Several studies have shown that the levels of expression of soluble guanylyl cyclase (sGC) or cGMP-dependent protein kinase (PKG) are altered in cultured VSMC. In this study the mechanisms involved in the coordinated expression of sGC and PKG were examined. Pro-inflammatory cytokines that increase the expression of type II NO synthase (inducible NO synthase, or iNOS) decreased PKG expression in freshly isolated, non-passaged bovine aortic SMC. However, in several passaged VSMC lines (i.e. bovine aortic SMC, human aortic SMC, and A7r5 cells), PKG protein expression was not suppressed by cytokines or NO. sGC was highly expressed in non-passaged bovine aortic SMC but not in passaged cell lines. Restoration of expression of sGC to passaged bovine SMC using adenovirus encoding the alpha1 and beta1 subunits of sGC restored the capacity of the cells to increase cGMP in response to NO. Furthermore, treatment of these sGC-transduced cells with NO donors for 48 h resulted in decreased PKG protein expression. In contrast, passaged rat aortic SMC expressed high levels of NO-responsive sGC but demonstrated reduced expression of PKG. Adenovirus-mediated expression of the PKG catalytically active domain in rat aortic SMC caused a reduction in the expression of sGC in these cells. These results suggest that there is a mechanism for the coordinated expression of sGC and PKG in VSMC and that prolonged activation of sGC down-regulates PKG expression. Likewise, the loss of PKG expression appears to increase sGC expression. These effects may be an adaptive mechanism allowing growth and survival of VSMC in vitro.     

POSTNATAL CHANGES IN THE LEVELS OF GLYCINE AND THE ACTIVITIES OF SERINE HYDROXYMETHYLTRANSFERASE AND GLYCINE: 2-OXOGLUTARATE AMINOTRANSFERASE IN THE RAT CENTRAL NERVOUS SYSTEM

Abstract— Of the amino acids found in the CNS of 10-day-old rats the concentration of glycine alone was significantly higher in the spinal cord than in all other regions. Spinal levels of glycine, cystathionine, isoleucine and lysine from 1- and 10-day-old rats did not differ significantly from adult values, whereas the levels of most other amino acids, including GABA, glutamate, glutamine and taurine, were higher in the young animals than in the adults. Aspartate was the only amino acid found in lower concentration in the spinal cord of young animals than in adult animals. These and other observations support the conclusion that glycine is used as an inhibitory transmitter in rat spinal cord early in postnatal life. There was a general decrease in the activity of serine hydroxymethyltransferase and a slight increase in the activity of glycine:2-oxoglutarate aminotransferase in the CNS during development. The activity of neither enzyme correlated on a regional basis with the glycine content. The high level of hydroxymethyltransferase activity in the cerebellum of 10-day-old rats suggests that the activity of this enzyme reflects cell growth rate.     

Nestin expression is lost in ventricular fibroblasts during postnatal development of the rat heart and re-expressed in scar myofibroblasts

Studies have reported that the intermediate filament protein nestin was expressed in various non-stem/progenitor cells during development, downregulated during postnatal growth and re-expressed following injury. The present study tested the hypothesis that an analogous paradigm was prevalent for ventricular fibroblasts. In the neonatal rat heart, nestin protein levels were significantly higher than the adult heart and the isolation of cardiac cells revealed a selective expression in ventricular fibroblasts. In adult ventricular fibroblasts, nestin protein expression was markedly lower compared to neonatal ventricular fibroblasts. Following ischemic damage to the rat heart, nestin staining was detected in a subpopulation of scar myofibroblasts (37%) and the percentage of immunoreactive cells was greater than adult ventricular fibroblasts (7%) but significantly lower than neonatal ventricular fibroblasts (86%). Moreover, dissimilar rates of (3)H-thymidine uptake were observed among the fibroblast populations and may be related in part to the disparate percentage of nestin(+) cells. To assess the role of nestin in DNA synthesis, neonatal ventricular fibroblasts were infected with a lentivirus containing a shRNAmir directed against the intermediate filament protein. The partial depletion of nestin expression in neonatal ventricular fibroblasts significantly reduced basal DNA synthesis, in the absence of an apoptotic response. Thus, postnatal development of the rat heart was associated with a selective loss of nestin expression in ventricular fibroblasts and subsequent induction in a subpopulation of myofibroblasts following ischemic injury. The re-expression of nestin in scar myofibroblasts may represent an adaptive response to enhance their proliferative rate and accelerate the healing process.     

Developmental changes of cytochrome c oxidase and citrate synthase in rat heart homogenate

Activity of cytochrome c oxidase and citrate synthase in rat heart homogenates was determined in 5-, 15-, 28- and 60-day-old rats. The activity of both enzymes increased during postnatal development but their changes followed different kinetics. The membrane-bound cytochrome c oxidase reached its adult values during the early postnatal period, i.e. between days 5 and 15, whereas soluble matrix-localized citrate synthase also continued to increase between days 15 and 60. Our data indicate a relative excess of cytochrome c oxidase in neonatal cardiocytes.     

Spontaneous and receptor-controlled soluble guanylyl cyclase activity in anterior pituitary cells

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.     

Effect of starvation on lipoprotein lipase activity in the liver of developing rats

Liver lipoprotein lipase activity in neonatal (1- and 5-day-old) rats was 2-3-times than in the liver of adult rats. In mid-suckling (15-day-old) or weaned (30-day-old) animals, it was not significantly different from the low activity detected in adult rats. Starvation resulted in a 3-fold increase of lipoprotein lipase activity in the neonatal liver, but did not affect the activity in the liver of mid-suckling, weaned or adult rats. When isolated livers from both 1- and 5-day-old pups were perfused with heparin, a sharp peak of lipoprotein lipase activity appeared in the perfusate. In fed neonates, the peak area accounted for about 70% of the total (released + non-releasable) activity. In starved neonates, the proportion of heparin-releasable activity increased up to about 90%. These results indicate that neonatal rat liver lipoprotein lipase activity is markedly affected by changes in the nutritional status of the animal, and the effect is restricted to the vascular pool of the enzyme, as was reported in extrahepatic tissues from adult rats.     

Overexpression of alpha 2-adrenergic receptors in fetal rat heart: receptors in search of a function.

alpha 2-Adrenergic receptors are transiently overexpressed by many types of developing cells. In the current study, the developmental profile and cellular function of these receptors were examined in the fetal and neonatal rat heart. alpha 2-Receptors, assessed with [3H]rauwolscine binding, were extremely high in fetal hearts on gestational day 19, 30-fold higher than values seen in adults. Receptor binding decreased by two-thirds by gestational day 21 and dropped by half again by postnatal day 3. To assess potential cellular functions controlled by the alpha 2-receptors, the capability of an alpha 2-agonist (clonidine) to inhibit membrane-associated adenylate cyclase activity was measured in three different settings: basal enzyme activity, the enzymatic response to isoproterenol (dependent upon beta-receptor linkages to the regulatory protein, Gs), and the response to forskolin (independent of receptor-Gs interactions). Despite the high number of alpha 2-receptors in fetal hearts, clonidine failed to alter any of the adenylate cyclase activity measures. In light of the postulated role of alpha 2-receptors in the maintenance of fetal/neonatal atrioventricular conduction, the excess alpha 2-receptors are probably linked to other cellular events, such as movement of calcium into the cell.     

The lipoprotein lipase (clearing-factor lipase) activity of cells isolated from rat cardiac muscle.

The total lipoprotein lipase activity recovered in suspension of cells prepared from adult rat hearts was unaffected by the nutritional state of the animals used. The enzyme activity present in the cell suspensions was almost exclusively associated with the cardiac muscle cells present as the major cell type.     

Effect of guanine nucleotides on polyphosphoinositide synthesis in rat liver plasma membranes.

The subcellular distributions of endogenous ADP-ribosylation products in hearts from 1-day-old neonatal and adult rats were investigated. In adult rat heart a 52 kDa mono-ADP-ribosylation product was identified in the plasma membrane fraction. In contrast, in neonatal rat heart a 130 kDa poly-ADP-ribosylation product was present in the nuclear fraction. The monomeric and polymeric nature of the two ADP-ribosylation products was determined by their sensitivity to thymidine and by analysis of their snake venom phosphodiesterase products. NADP+ enhanced both the mono- and polymeric reactions. The ADP-ribose-protein linkage of the adult 52 kDa product was stable to 1 h of treatment with hydroxylamine (0.5 M) and mercury ions, but was sensitive to alkali and a 12 h treatment with hydroxylamine (1 M). This is suggestive of an arginine linkage. The 130 kDa poly-ADP-ribosylation product from the neonatal rat heart was alkalilabile but stable to both hydroxylamine and HgCl2. This implies the presence of an unusual linkage in the 130 kDa product. The presence of these different ADP-ribosylation products in adult and neonatal rat hearts suggests the possible importance of these proteins and their ADP-ribosylation during cardiac development.     

Impaired iNOS–sGC–cGMP signalling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS–sGC–cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague–Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight‐matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle + septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α₁ mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension. Copyright © 2012 John Wiley & Sons, Ltd.     

Cell-specific expression and regulation of soluble guanylyl cyclase alpha 1 and beta 1 subunits in the rat ovary

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC alpha(1) and beta(1) subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC alpha(1) subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC alpha(1) immunoreactivity. In gonadotropin- treated immature rats, intense sGC alpha(1) subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC alpha(1) immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC beta(1), indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC alpha(1) and beta(1) subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.     

Impaired iNOS-sGC-cGMP signalling contributes to chronic hypoxic and hypercapnic pulmonary hypertension in rat

Nitric oxide (NO) is an important vascular modulator in the development of pulmonary hypertension. NO exerts its regulatory effect mainly by activating soluble guanylate cyclase (sGC) to synthesize cyclic guanosine monophosphate (cGMP). Exposure to hypoxia causes pulmonary hypertension. But in lung disease, hypoxia is commonly accompanied by hypercapnia. The aim of this study was to examine the changes of sGC enzyme activity and cGMP content in lung tissue, as well as the expression of inducible nitric oxide synthase (iNOS) and sGC in rat pulmonary artery after exposure to hypoxia and hypercapnia, and assess the role of iNOS-sGC-cGMP signal pathway in the development of hypoxic and hypercapnic pulmonary hypertension. Male Sprague-Dawley rats were exposed to hypoxia and hypercapnia for 4 weeks to establish model of chronic pulmonary hypertension. Weight-matched rats exposed to normoxia served as control. After exposure to hypoxia and hypercapnia, mean pulmonary artery pressure, the ratio of right ventricle/left ventricle+septum, and the ratio of right ventricle/body weight were significantly increased. iNOS mRNA and protein levels were significantly increased, but sGC α(1) mRNA and protein levels were significantly decreased in small pulmonary arteries of hypoxic and hypercapnic exposed rat. In addition, basal and stimulated sGC enzyme activity and cGMP content in lung tissue were significantly lower after exposure to hypoxia and hypercapnia. These results demonstrate that hypoxia and hypercapnia lead to the upregulation of iNOS expression, downregulation of sGC expression and activity, which then contribute to the development of pulmonary hypertension.     

Cyclin I and p53 are differentially expressed during the terminal differentiation of the postnatal mouse heart

In this study, we have used Ki-67 and MF20 mAb to determine how extensively cardiomyocytes proliferate in the postnatal mouse heart. It was established that the cardiomyocytes divided rapidly in 2-day-old hearts. However, at 13 days, the majority of cardiomyocytes had entered into terminal growth arrest and differentiation. We exploited this finding in order to identify proteins that were associated with cardiomyocyte growth and differentiation. The protein profiles of 2- and 13-day-old hearts were established by two-dimensional electrophoresis and compared. Seventeen protein spots were found to be differentially expressed at day 13. Eight of them were up-regulated while the remaining nine protein spots were down-regulated. We focused our attention on 2 of the proteins identified by MALDI-TOF MS, cyclin I and p53, because they are both believed to be involved in cell cycle regulation. Western blot analysis confirmed that both proteins were positively up-regulated in the 13-day-old postnatal heart. To determine directly whether these proteins were associated with cell proliferation, we examined their expression patterns in H9c2 cardiomyocytes maintained in vitro. We established that cyclin I expression was low during the growing phase of H9c2 culture and high during the growth arrest/differentiation phases. In contrast, p53 expression was unchanged during both phases. The various growth phases were confirmed by the presence of cyclin A and growth arrest-specific 1 proteins. We investigated whether silencing cyclin I expression using cyclin I-siRNA could promote an increase in H9c2 cell proliferation. It was determined that silencing cyclin I could enhance a small, but significant, increase in H9c2 cell division. Similar results were obtained for cardiomyocytes extracted from 13-day-old hearts. These results imply that the reason why cardiomyocytes in 13-day-old hearts increased cyclin I expression was probably associated with terminal growth arrest. However, the increase in p53 expression was probably associated with cardiomyocyte differentiation, rather than growth arrest.