Robert C. Fowkes

Regulation of cardiovascular cell function by hydrogen sulfide (H2S)

Since the discovery of endogenously‐produced hydrogen sulfide (H2S) in various tissues, there has been an explosion of interest in H2S as a biological mediator alongside other gaseous mediators, nitric oxide and carbon monoxide. The identification of enzyme‐regulated H2S synthetic pathways in the cardiovascular system has led to a number of studies examining specific regulatory actions of H2S. We review evidence showing that endogenously‐generated and exogenously‐administered H2S exerts a wide range of actions in vascular and myocardial cells including vasodilator/vasoconstrictor effects via modification of the smooth muscle tone, induction of apoptosis and anti‐proliferative responses in the smooth muscle cells, angiogenic actions, effects relevant to inflammation and shock, and cytoprotection in models of myocardial ischemia‐reperfusion injury. Several molecular mechanisms of action of H2S have been described. These include interactions of H2S with NO, redox regulation of multiple signaling proteins and regulation of KATP channel opening. The gaps in our current understanding of precise mechanisms, the absence of selective pharmacological tools and the limited availability of H2S measurement techniques for living tissues, leave many questions about physiological and pathophysiological roles of H2S unanswered at present. Nevertheless, this area of investigation is advancing rapidly. We believe H2S holds promise as an endogenous mediator controlling a wide range of cardiovascular cell functions and integrated responses under both physiological and pathological conditions and may be amenable to therapeutic manipulation. Copyright

A maternal "junk food" diet in pregnancy and lactation promotes nonalcoholic Fatty liver disease in rat offspring.

With rising obesity rates, nonalcoholic fatty liver disease is predicted to become the main cause of chronic liver disease in the next decades. Rising obesity prevalence is attributed to changes in dietary habits with increased consumption of palatable junk foods, but maternal malnutrition also contributes to obesity in progeny. This study examines whether a maternal junk food diet predisposes offspring to nonalcoholic fatty liver disease. The 144 rat offspring were fed either a balanced chow diet alone or with palatable junk foods rich in energy, fat, sugar, and/or salt during gestation, lactation, and/or after weaning up to the end of adolescence. Offspring fed junk food throughout the study exhibited exacerbated hepatic steatosis, hepatocyte ballooning, and oxidative stress response compared with offspring given free access to junk food after weaning only. These offspring also displayed sex differences in their hepatic molecular metabolic adaptation to diet-induced obesity with increased expression of genes associated with insulin sensitivity, de novo lipogenesis, lipid oxidation, and antiinflammatory properties in males, whereas the gene expression profile in females was indicative of hepatic insulin resistance. Hepatic inflammation and fibrosis were not detected indicating that offspring had not developed severe steatohepatitis by the end of adolescence. Hepatic steatosis and increased oxidative stress response also occurred in offspring born to junk food-fed mothers switched to a balanced chow diet from weaning, highlighting a degree of irreversibility. This study shows that a maternal junk food diet in pregnancy and lactation contributes to the development of nonalcoholic fatty liver disease in offspring.

Somatostatin analogs modulate AIP in somatotroph adenomas: the role of the ZAC1 pathway.

CONTEXT Somatotroph adenomas harboring aryl hydrocarbon receptor interacting protein (AIP) mutations respond less well to somatostatin analogs, suggesting that the effects of somatostatin analogs may be mediated by AIP. OBJECTIVE The objective of the investigation was to study the involvement of AIP in the mechanism of effect of somatostatin analogs. DESIGN In the human study, a 16-wk somatostatin analog pretreatment compared with no pretreatment. In the in vitro cell line study, the effect of somatostatin analog treatment or small interfering RNA (siRNA)/plasmid transfection were studied. SETTING The study was conducted at a university hospital. PATIENTS Thirty-nine sporadic and 10 familial acromegaly patients participated in the study. INTERVENTION Interventions included preoperative lanreotide treatment and pituitary surgery. OUTCOME For the human study, GH and IGF-I levels, AIP, and somatostatin receptor staining were measured. For the cell line, AIP and ZAC1 (zinc finger regulator of apoptosis and cell cycle arrest) expression, metabolic activity, and clone formation were measured. RESULTS Lanreotide pretreatment reduced GH and IGF-I levels and tumor volume (all P < 0.0001). AIP immunostaining was stronger in the lanreotide-pretreated group vs. the surgery-only group (P < 0.001). After lanreotide pretreatment, the AIP score correlated to IGF-I changes in females (R = 0.68, P < 0.05). Somatostatin receptor staining was not reduced in samples with AIP mutations. In GH3 cells, 1 nm octreotide increased AIP mRNA and protein (both P < 0.01) and ZAC1 mRNA expression (P < 0.05). Overexpression of wild-type (but not mutant) AIP increased ZAC1 mRNA expression, whereas AIP siRNA knockdown reduced ZAC1 mRNA (both P < 0.05). The siRNA-mediated knockdown of AIP led to an increased metabolic activity and clonogenic ability of GH3 cells compared with cells transfected with a nontargeting control (both P < 0.001). CONCLUSION These results suggest that AIP may play a role in the mechanism of action of somatostatin analogs via ZAC1 in sporadic somatotroph tumors and may explain their lack of effectiveness in patients with AIP mutations.

C-type natriuretic peptide: an important neuroendocrine regulator?

In the decade since its discovery, C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been shown to be produced by most of the major endocrine glands, including the hypothalamus and anterior pituitary. The relative abundance of its guanylyl cyclase-containing GC-B receptor in these glands suggests that CNP might be a local neuroendocrine regulator. Here, we review this possibility, emphasizing signalling and integration with other regulatory systems in the neuroendocrine control of reproduction.

Pulsatile and sustained gonadotropin-releasing hormone (GnRH) receptor signaling: Does the Ca2+/NFAT signaling pathway decode GnRH pulse frequency?

Gonadotropin-releasing hormone (GnRH) acts via 7 transmembrane region receptors on gonadotrophs to stimulate synthesis and secretion of the luteinizing hormone and follicle-stimulating hormone. It is secreted in pulses, and its effects depend on pulse frequency, but decoding mechanisms are unknown. Here we have used (nuclear factor of activated T-cells 2 (NFAT2)-emerald fluorescent protein) to monitor GnRH signaling. Increasing [Ca2+]i causes calmodulin/calcineurin-dependent nuclear NFAT translocation, a response involving proteins (calmodulins and NFATs) that decode frequency in other systems. Using live cell imaging, pulsatile GnRH caused dose- and frequency-dependent increases in nuclear NFAT2-emerald fluorescent protein, and at low frequency, translocation simply tracked GnRH exposure (albeit with slower kinetics). At high frequency (30-min intervals), failure to return to basal conditions before repeat stimulation caused integrative tracking, illustrating how the relative dynamics of up- and downstream signals can increase efficiency of GnRH action. Mathematical modeling predicted desensitization of GnRH effects on [Ca2+]i and that desensitization would increase with dose, frequency, and receptor number, but no such desensitization was seen in HeLa and/or LβT2 cells possibly because pulsatile GnRH did not reduce receptor expression (measured by immunofluorescence). GnRH also caused dose- and frequency-dependent activation of αGSU, luteinizing hormone β, and follicle-stimulating hormone β luciferase reporters, effects that were blocked by calcineurin inhibition. Pulsatile GnRH also activated an NFAT-responsive luciferase reporter, but this response was directly related to cumulative pulse duration. This together with the lack of desensitization of translocation responses suggests that NFAT may mediate GnRH action but is not a genuine decoder of GnRH pulse frequency.

Regulation of Human Glycoprotein Hormone α-Subunit Gene Transcription in LβT2 Gonadotropes by Protein Kinase C and Extracellular Signal-Regulated Kinase 1/2

Abstract Transcriptional activation of the human glycoprotein hormone α-subunit (αGSU) promoter in response to GnRH and phorbol-12-myristate-13-acetate (PMA) has been well characterized in αT3-1 gonadotropes but not investigated in the more differentiated LβT2 clonal gonadotrope. We have evaluated αGSU transcription in the more mature LβT2 cell line, using deletion and heterologous constructs of the αGSU promoter linked to a luciferase reporter gene. Basal αGSU-promoter activity was significantly less in LβT2 cells than in αT3-1 cells, but stimulation of transfected cells with GnRH and PMA resulted in similar increases in αGSU-promoter activity. Deletional analysis of the human αGSU promoter in LβT2 cells indicated that sequences between −398 and −244 and between −244 and −195 base pairs (bp) were involved in regulating basal αGSU-promoter transcription, whereas the region between −244 and −195 bp regulated PMA-stimulated promoter activity. Deletion of this promoter region containing a steroidogenic factor-1 (SF-1) binding site abolished basal and PMA-stimulated transcription. Site-directed mutagenesis of the SF-1 binding site resulted in a significant attenuation of basal and PMA-stimulated αGSU transcription. Pretreatment of LβT2 cells with a mitogen-activated protein kinase kinase-specific inhibitor, U0126, abolished the PMA-stimulated increase in MAPK activity and significantly reduced basal and PMA-stimulated promoter activity. Electrophoretic mobility shift assays for SF-1 and GATA revealed that PMA failed to affect SF-1 binding but enhanced GATA binding to a consensus GATA oligonucleotide, an effect that was blocked with U0126 pretreatment, suggesting that GATA may mediate ERK activation of αGSU transcription. Our data suggests that, in the mature LβT2 gonadotrope cell line, two regions of the human αGSU promoter regulate basal transcription and that SF-1 is involved in mediating basal and PMA-stimulated promoter activity. Furthermore, PKC-stimulated transcription partially relies on ERK acting on elements downstream of −244 bp of the human αGSU promoter.

L-Cysteine Stimulates Hydrogen Sulfide Synthesis in Myocardium Associated With Attenuation of Ischemia-Reperfusion Injury:

Hydrogen sulfide (H 2S) is a biological mediator produced by enzyme-regulated pathways from L-cysteine, which is a substrate for cystathionine-γ-lyase (CSE). In myocardium, endogenously and exogenously administered H2S has been shown to protect against ischemia-reperfusion injury. We hypothesized that L-cysteine exerts its protective action through stimulation of H2S production. Rat isolated hearts were Langendorff-perfused and underwent 35-minute regional ischemia and 120-minute reperfusion. L-cysteine perfusion from 10 minutes before ischemia until 10 minutes after reperfusion limited infarct size in a concentration-dependent manner, maximal at 1 mmol/L (control 36.4% ± 2.4% vs L-cysteine 24.3% ± 3.4%, P < .05). This protective action was attenuated by the CSE inhibitor, DL-propargylglycine (PAG) 1 mmol/L (31.4 ± 5.9%, not significant vs control) but administration of the CSE cofactor pyridoxal-5′-phosphate (PLP) 50 μmol/L did not enhance the effect of L-cysteine. Ten minutes normoxic perfusion with L-cysteine 1 mmol/L caused a 3-fold increase in myocardial H2S concentration (0.64 ± 0.16 vs 2.01 ± 0.07 μmol/g protein, P < .01), an effect that was significantly attenuated by PAG (1.17 ± 0.15 μmol/g protein). These data provide evidence that exogenous L-cysteine administration limits ischemia-reperfusion injury through a mechanism that appears to be at least partially dependent on H2S synthesis.

Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, αT3-1 and LβT2 gonadotroph cells

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both alphaT3-1 and LbetaT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in alphaT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone alpha-subunit promoter in LbetaT2 cells but not in alphaT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.

Suppression of nuclear factor-κB activity in macrophages by chylomicron remnants: modulation by the fatty acid composition of the particles.

Current evidence indicates that chylomicron remnants (CMR) induce macrophage foam cell formation, an early event in atherosclerosis. Inflammation also plays a part in atherogenesis and the transcription factor nuclear factor‐κB (NF‐κB) has been implicated. In this study, the influence of CMR on the activity of NF‐κB in macrophages and its modulation by the fatty acid composition of the particles were investigated using macrophages derived from the human monocyte cell line THP‐1 and CMR‐like particles (CRLPs). Incubation of THP‐1 macrophages with CRLPs caused decreased NF‐κB activation and downregulated the expression of phospho‐p65–NF‐κB and phospho‐IκBα (pIκBα). Secretion of the inflammatory cytokines tumour necrosis factor α, interleukin‐6 and monocyte chemoattractant protein‐1, which are under NF‐κB transcriptional control, was inhibited and mRNA expression for cyclooxygenase‐2, an NF‐κB target gene, was reduced. CRLPs enriched in polyunsaturated fatty acids compared with saturated or monounsaturated fatty acids had a markedly greater inhibitory effect on NF‐κB binding to DNA and the expression of phospho‐p65–NF‐κB and pIκB. Lipid loading of macrophages with CRLPs enriched in polyunsaturated fatty acids compared with monounsaturated fatty acids or saturated fatty acids also increased the subsequent rate of cholesterol efflux, an effect which may be linked to the inhibition of NF‐κB activity. These findings demonstrate that CMR suppress NF‐κB activity in macrophages, and that this effect is modulated by their fatty acid composition. This downregulation of inflammatory processes in macrophages may represent a protective effect of CMR which is enhanced by dietary polyunsaturated fatty acids.

11β-Hydroxysteroid dehydrogenase enzymes in the testis and male reproductive tract of the boar (Sus scrofa domestica) indicate local roles for glucocorticoids in male reproductive physiology

11Beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes modulate the target cell actions of corticosteroids by catalysing metabolism of the physiological glucocorticoid (GC), cortisol, to inert cortisone. Recent studies have implicated GCs in boar sperm apoptosis. Hence, the objective of this study was to characterise 11betaHSD enzyme expression and activities in the boar testis and reproductive tract. Although 11betaHSD1 and 11betaHSD2 mRNA transcripts and proteins were co-expressed in all tissues, cortisol-cortisone interconversion was undetectable in the corpus and cauda epididymides, vas deferens, vesicular and prostate glands, irrespective of nucleotide cofactors. In contrast, homogenates of boar testis, caput epididymidis and bulbourethral gland all displayed pronounced 11betaHSD activities in the presence of NADPH/NADP(+) and NAD(+), and the penile urethra exhibited NAD(+)-dependent 11beta-dehydrogenase activity. In kinetic studies, homogenates of boar testis, caput epididymidis and bulbourethral gland oxidised cortisol with K(m) values of 237-443 and 154-226 nmol/l in the presence of NADP(+) and NAD(+) respectively. Maximal rates of NADP(+)-dependent cortisol oxidation were 7.4- to 28.5-fold greater than the V(max) for NADPH- dependent reduction of cortisone, but were comparable with the rates of NAD(+)-dependent cortisol metabolism. The relatively low K(m) estimates for NADP(+) -dependent cortisol oxidation suggest that either the affinity of 11betaHSD1 has been increased or the cortisol inactivation is catalysed by a novel NADP(+)-dependent 11betaHSD enzyme in these tissues. We conclude that in the boar testis, caput epididymidis and bulbourethral gland, NADP(+)- and NAD(+)-dependent 11betaHSD enzymes catalyse net inactivation of cortisol, consistent with a physiological role in limiting any local actions of GCs within these reproductive tissues.