Differential contribution of nitric oxide and cGMP to the stimulatory effects of growth hormone-releasing hormone and low-concentration somatostatin on growth hormone release from somatotrophs

There is increasing evidence that nitric oxide (NO) produced by NO synthase (NOS), and their signalling partners, guanylyl cyclase and cGMP, play a relevant role in growth hormone (GH) secretion from somatotrophs. We previously demonstrated that both GH-releasing hormone (GHRH; 10(-8) M) and low concentrations of somatostatin (10(-15) M) stimulate pig GH release in vitro, whereas a high somatostatin concentration (10(-7) M) inhibits GHRH-induced GH secretion. To ascertain the possible contribution of the NOS-NO and guanylyl cyclase-cGMP routes to these responses, cultures of pituitary cells from prepubertal female pigs were treated (30 min) with GHRH (10(-8) M) or somatostatin (10(-7) or 10(-15) M) in the absence or presence of activators or blockers of key steps of these signalling cascades, and GH release was measured. Two distinct activators of NO route, SNAP (5x10(-4) M) or L-AME (10(-3) M), similarly stimulated GH release when applied alone (with this effect being blocked by 10(-7) M somatostatin), but did not alter the stimulatory effect of GHRH or 10(-15) M somatostatin. Conversely, two NO pathway inhibitors, NAME (10(-5) M) or haemoglobin (20 microg/ml) similarly blocked GHRH- or 10(-15) M somatostatin-stimulated GH release. 8-Br-cGMP (10(-8) to 10(-4) M) strongly stimulated GH release, suggesting that cGMP may function as a subsequent step in the NO pathway in this system. Interestingly, 10(-7) M somatostatin did not inhibit the stimulatory effect of 8-Br-cGMP. Moreover, although 8-Br-cGMP did not modify the effect of GHRH, it enhanced GH release stimulated by 10(-15) M somatostatin. Accordingly, a specific guanylyl cyclase inhibitor, LY-83, 583 (10(-5) M) did not alter 10(-15) M somatostatin-induced GH release, whereas it blocked GHRH-induced GH secretion. These results demonstrate for the first time that the NOS/NO signalling pathway contributes critically to the stimulatory effects of both GHRH and low-concentration somatostatin on GH release, and that, conversely, the subsequent guanylyl cyclase/cGMP step only mediates GHRH- and not low-concentration somatostatin-induced GH secretion from somatotrophs.     

A G alpha-dependent pathway that antagonizes multiple chemoattractant responses that regulate directional cell movement

Chemotactic cells, including neutrophils and Dictyostelium discoideum, orient and move directionally in very shallow chemical gradients. As cells polarize, distinct structural and signaling components become spatially constrained to the leading edge or rear of the cell. It has been suggested that complex feedback loops that function downstream of receptor signaling integrate activating and inhibiting pathways to establish cell polarity within such gradients. Much effort has focused on defining activating pathways, whereas inhibitory networks have remained largely unexplored. We have identified a novel signaling function in Dictyostelium involving a Galpha subunit (Galpha9) that antagonizes broad chemotactic response. Mechanistically, Galpha9 functions rapidly following receptor stimulation to negatively regulate PI3K/PTEN, adenylyl cyclase, and guanylyl cyclase pathways. The coordinated activation of these pathways is required to establish the asymmetric mobilization of actin and myosin that typifies polarity and ultimately directs chemotaxis. Most dramatically, cells lacking Galpha9 have extended PI(3,4,5)P(3), cAMP, and cGMP responses and are hyperpolarized. In contrast, cells expressing constitutively activated Galpha9 exhibit a reciprocal phenotype. Their second message pathways are attenuated, and they have lost the ability to suppress lateral pseudopod formation. Potentially, functionally similar Galpha-mediated inhibitory signaling may exist in other eukaryotic cells to regulate chemoattractant response.     

Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships

The structure-activity relationships of flavonoids with regard to their inhibitory effects on phosphodiesterase (PDE) isozymes are little known. The activities of PDE1-5 were measured by a two-step procedure using cAMP with [(3)H]-cAMP or cGMP with [(3)H]-cGMP as substrates. In the present results, PDE1, 5, 2, and 4 isozymes were partially purified from guinea pig lungs in that order, and PDE3 was from the heart. The IC(50) values of PDE1-5 were greater than those reported previously for the reference drugs, vinpocetin, EHNA, milrinone, Ro 20-1724, and zaprinast, by 5-, 5-, 7-, 5-, and 3-fold, respectively. As shown in Table 2, luteolin revealed non-selective inhibition of PDE1-5 with IC(50) values in a range of 10-20 microM, as did genistein except with a low potency on PDE5. Daidzein, an inactive analogue of genistein in tyrosine kinase inhibition, showed selective inhibition of PDE3 with an IC(50) value of around 30 microM, as did eriodictyol with an IC(50) value of around 50 microM. Hesperetin and prunetin exhibited more-selective inhibition of PDE4 with IC(50) values of around 30 and 60 microM, respectively. Luteolin-7-glucoside exhibited dual inhibition of PDE2/PDE4 with an IC(50) value of around 40 microM. Diosmetin more-selectively inhibited PDE2 (IC(50) of 4.8 microM) than PDE1, PDE4, or PDE5. However, biochanin A more-selectively inhibited PDE4 (IC(50) of 8.5 microM) than PDE1 or PDE2. Apigenin inhibited PDE1-3 with IC(50) values of around 10-25 microM. Myricetin inhibited PDE1-4 with IC(50) values of around 10-40 microM. The same was true for quercetin, but we rather consider that it more-selectively inhibited PDE3 and PDE4 (IC(50) of < 10 microM). In conclusion, it is possible to synthesize useful drugs through elucidating the structure-activity relationships of flavonoids with respect to inhibition of PDE isozymes at concentrations used in this in vitro study.     

Cytokines, nitric oxide, and cGMP modulate the permeability of an in vitro model of the human blood-brain barrier

The endothelial cells (EC) of the microvasculature in the brain form the anatomical basis of the blood-brain barrier (BBB). In the present study, the effects of agents that modify the permeability of a well-established in vitro model of the human BBB were studied. The monolayers formed by confluent human brain microvessel endothelial cell (HBMEC) cultures are impermeable to the macromolecule tracer horseradish peroxidase (HRP) and have high electrical resistance. Exposure of HBMEC to various cytokines including TNF-alpha, IL-1beta, interferon gamma (IFN-gamma), or lipopolysaccharide (LPS) decreased transendothelial electrical resistance (TEER) mainly by increasing the permeability of the tight junctions. Primary cultures of HBMEC express endothelial nitric oxide synthase (eNOS) and produce low levels of NO. Treatment with the NO donors sodium nitroprusside (SNP) and DETA NONOate or the cGMP agonist 8-Br-cGMP significantly increased monolayer resistance. Conversely, inhibition of soluble guanylyl cyclase with ODQ rapidly decreased the resistance, and pretreatment of HBMEC with Rp-8-CPT-cGMPS, an inhibitor of cGMP-dependent protein kinase, partially prevented the 8-Br-cGMP-induced increase in resistance. Furthermore, NO donors and 8-Br-cGMP could also reverse the increased permeability of the monolayers induced by IL-1beta, IFN-gamma, and LPS. These results indicate that NO can decrease the permeability of the human BBB through a mechanism at least partly dependent on cGMP production and cGMP-dependent protein kinase activation.     

脱氢紫堇碱体外对兔血小板中环核苷酸含量的影响

目的研究脱氢紫堇碱(DHC)对兔血小板中cAMP 和cGMP含量的影响以探讨其抑制血小板聚集的作用机制.方法应用放射免疫分析方法.结果DHC显著增加兔血小板中cAMP的含量,而降低兔血小板中cGMP的含量.结论DHC可能通过增加血小板中cAMP的含量和降低血小板中cGMP的含量而产生血小板抑制作用.     

吗啡依赖大鼠脊髓和脑干一氧化氮含量和合酶活力分析

本文检测了吗啡依赖大鼠脊髓和脑干一氧化氮合酶（ＮＯｓ）活力,一氧化氮（ＮＯ）以及ｃＧＭＰ含量。结果显示吗啡依赖大鼠脊髓ＮＯｓ活力,ＮＯ和ｃＧＭＰ含量较正常对照组降低,脑干中ＮＯｓ活力轻度降低,而ＮＯ和ｃＧＭＰ含量降低。纳洛酮激发戒断症状后大鼠脊髓和脑干ＮＯｓ活力,ＮＯ以及ｃＧＭＰ含量急剧升高。ＮＯｓ抑制剂Ｌ－Ｎ－硝基精氨酸甲酯（Ｌ－ＮＡＭＥ）处理可以抑制大鼠吗啡戒断反应,同时减少脊髓和脑干ＮＯ含量。结果表明吗啡戒断反应与脊髓和脑干的ＮＯ－ｃＧＭＰ途径的兴奋有关。     

Pharmacotherapy for erectile dysfunction

Erectile dysfunction (ED) is defined as the consistent inability to obtain or maintain an erection for satisfactory sexual relations. An estimated 20–30 million men suffer from some degree of sexual dysfunction. The past 20 years of research on erectile physiology have increased our understanding of the biochemical factors and intracellular mechanisms responsible for corpus cavernosal smooth muscle contraction and relaxation, and revealed that ED is predominantly a disease of vascular origin. Since the advent of sildenafil (Viagra®), there has been a resurgence of interest in ED, and an increase in patients presenting with this disease. A thorough knowledge of the physiology of erection is essential for future pharmacological innovations in the field of male ED.     

cAMP and cGMP in the human parotid saliva

Summary Human parotid saliva was revealed to contain considerable amounts of cAMP and cGMP. On sour lemon drop stimulation, the salivary cyclic nucleotides concentration decreased and the cyclic nucleotides secretory rate increased in close relationship with the salivary flow rate in control subjects. On feeding, both cAMP concentration and secretory rate increased significantly in control subjects. Parotid saliva from a patient of Sjögren's syndrom was revealed to have a significantly higher cGMP concentration than those from control subjects. Saliva derived from the malignant tumor of the parotid gland was revealed to have a higher cGMP concentration and a lower cAMP concentration than the healthy site. From these findings, the possibilities of the clinical usefulness of studying salivary cyclic nucleotides were discussed.     

New advances in models and strategies for developing anti-obesity drugs

Introduction: Obesity is a worldwide pandemic. Obesity-related health and economic costs are staggering. Existing strategies to combat obesity through lifestyle improvements and medical intervention have had limited success. Pharmacotherapy, in combination with lifestyle modification, may play a vital role in reversing the disease burden. However, past and current weight-loss medications have had serious safety risks, notably cardiovascular and psychiatric events.

Areas covered: The authors review the strategies for designing new anti-obesity drugs by describing those currently in development. They describe their target, mechanism of action and developmental or regulatory status. Furthermore, they discuss the problem of weight regain following weight loss, and its relevance to the long-term success of anti-obesity pharmacotherapy.

Expert opinion: For weight management drugs to achieve the safety and efficacy required to be impactful, current studies are uncovering and characterizing new targets, including new signaling circuits and hormones regulating appetite and metabolism, and re-evaluating the role of pharmacotherapy in weight management. To avoid the safety failures of many past weight-loss drugs, the models and strategies covered in this article incorporate recent advances in knowledge and technology. We discuss the emergence of cGMP signaling as a potentially transformative target in weight management. Modulating cGMP signaling may represent an ideal goal for an anti-obesity pharmacotherapy, reflecting some of the major themes described in the present review: targeting pathways that are newly realized as relevant for weight management; promoting safety by re-purposing drugs that are safe, proven, and approved for clinical use; and having a synergistic effect on multiple, reinforcing pathways.     

PKC抑制剂灯盏花素乙对内脏炎症痛大鼠脊髓NO/cGMP信号转导系统的影响

目的探讨灯盏花素乙在甲醛复制的内脏炎症痛中的作用及其对NO/cGMP信号转导系统的影响。方法成年健康Wistar大鼠,随机分为4组:正常对照组、内脏炎症痛组、溶媒组和灯盏花素乙组。观察:①鞘内注射灯盏花素乙后大鼠行为学变化,以15min为一个时间段,共2h,计算疼痛分数;②致痛后30min取脊髓,用分光光度计法测定NOS活性、NO产量和放射免疫法测定cGMP含量。结果①灯盏花素乙组在前90min内疼痛分数低于内脏炎症痛组(P<0.05orP<0.01);②内脏炎症痛、溶媒和灯盏花素乙组脊髓的NOS活性、NO产量和cGMP含量均高于正常对照组(P<0.05orP<0.01),灯盏花素乙组低于内脏炎症痛组(P<0.01)。结论①灯盏花素乙能减轻甲醛复制的内脏炎症痛;②灯盏花素乙可能通过抑制脊髓内PKC的激活,进而抑制NO/cGMP信号转导系统的激活。