Differential regulation on human skin fibroblast by alpha1 adrenergic receptor subtypes

Alpha 1 adrenoceptor (alpha1-AR) regulation of DNA synthesis was studied in human neonatal foreskin fibroblast. Saturation assay with a specific radioligand for alpha1 adrenergic [3H]-prazosin revealed two saturated and specific binding sites with high or low affinity. Competitive binding assay with different antagonist subtypes, defined pharmacologically three major types of alpha1-AR. The alpha1-AR agonists (from 1x10(-10) to 1x10(-4) M) triggered a biphasic action on DNA synthesis reaching maximal stimulation at 1x10(-9) M and maximal inhibition at 1x10(-6) M. Prazosin, abolished the stimulatory (pA2: 9.24) and inhibitory (pA2: 8.80) actions of alpha1-AR agonists. The alpha1-AR stimulation resulted in the activation of phosphoinositide turnover (InsP) via phospholipase C (PLC) involving calcium/calmodulin (CaM) and nitric oxide synthase (NOS) that correlates with the DNA synthesis increment; whereas the inhibition resulted in a decrease of cyclic AMP (cAMP) accumulation via adenylate cyclase inhibition. The potency displayed by the specific antagonists tested in binding, DNA synthesis, InsP and NOS at low agonist concentration suggests that they can be elicited by the activation of the same receptor (alpha1B-AR subtype); while the decrement in DNA synthesis and cAMP at high concentration account by the activation of alpha1D-AR coupled to Gi protein. Non-functional alpha1A-AR in neonatal human foreskin fibroblast was observed. Results suggest that the expression of alpha1-AR subtypes on human skin fibroblast may differentially activate signaling pathways that modulate physiological response of the cells.     

黄芪舒张血管平滑肌的作用及机制

目的:探讨黄芪对血管平滑肌的舒张作用及其机制.方法:采用大鼠离体胸主动脉环灌流模型.在去除血管环内皮后,观察累积浓度黄芪对基础状态(基础组)、苯肾上腺素(PE)预收缩(苯肾上腺素组)、氯化钾预收缩(氯化钾组)的血管环的作用和黄芪对经无钙液(无钙液组)、无钙液加肝素(肝素组)、普萘洛尔(普萘洛尔组)预处理后的血管环的作用.结果:在血管内皮被去除后,黄芪对基础状态或氯化钾预收缩的血管环无影响;当黄芪浓度累积达(10-1、3×10-1、100、3×100)g/L时,对PE(3×10-7mol/L)预收缩的血管环产生剂量依赖性舒张作用(P<0.05).经无钙液预处理后,黄芪(3×100 g/L)对血管环的舒张作用未被阻断;但经无钙液加肝素预处理后,该作用被显著抑制(P<0.01);而普萘洛尔预处理不影响黄芪对PE预收缩血管环的舒张作用.结论:黄芪对去除内皮的血管具有舒张作用.其机制可能与阻断血管平滑肌细胞内质网上的三磷酸肌醇敏感的钙离子通道,抑制内钙的释放有关.     

Signal transduction pathways associated with ATP-induced proliferation of cell progenitors in the intact embryonic retina

ATP and ADP induce retinal cell proliferation through activation of PKC and extracellular signal-regulated kinases (ERKs). Here, we characterized the effect of purinergic agonists on the turnover of phosphoinositides and activation of ERKs during development of the chick embryo retina. When intact retinas were incubated with ATP, ADP or UTP, a dose-dependent accumulation of [³H]-phosphoinositides was observed (% of control, EC₅₀: 548 ± 20.5%, 0.18 mM; 314 ± 53.8%, 0.51 mM; 704 ± 139.9%, 0.018 mM, respectively). Only the response promoted by ADP was completely inhibited by the P2 receptor antagonists, PPADS and suramin. All the responses decreased with the progression of retinal development. Western blot assays revealed that ATP, ADP and UTP stimulated the phosphorylation of ERKs in the chick embryo retina very early during development (% of control: 174 ± 16; 199 ± 16.4 and 206 ± 37, respectively). The responses to ADP and UTP were transient and dose-dependent, showing EC₅₀ values of 0.12 mM and 0.009 mM. The response to ADP was inhibited by the antagonists PPADS and suramin and by U73122 and chelerythrine chloride, which block PLC and PKC, respectively. Conversely, chelerythrine chloride did not block the response induced by UTP. Immunohistochemical analysis revealed that ATP and ADP induced the phosphorylation of ERKs in cells of the neuroblastic layer of retinas from embryos at E8. Our data showed that ATP, ADP and UTP stimulate the turnover of InsPs and promoted the activation of ERKs in the chick embryo retina. ADP, through activation of P2Y₁ receptors, activated ERK pathway through PLC and PKC and UTP, via P2Y₄-like receptors, induced the phosphorylation of ERKs through a pathway that did not involve PKC.     

Depolarisation of guinea-pig visceral smooth muscle causes hydrolysis of inositol phospholipids

Summary Increasing concentrations of KCl caused a progressive stimulation of contractile activity in guinea-pig jejunal longitudinal smooth muscle strips, accompanied by increased production of [³H]inositol phosphates in smooth muscle fragments pre-labelled with myo-[³H]inositol. The concentration-response curve for contractility lay to the left of that for [³H]inositol phosphate production. Both responses showed a dependency on the presence of Ca²⁺ in the incubation medium. K⁺-induced contractility was abolished by D600 or by Mn²⁺, whereas stimulated [³H]inositol phosphate formation persisted in the presence of these Ca²⁺-channel blockers. The simultaneous addition of high KCl concentrations together with a maximal concentration of neurotransmitter (carbamylcholine or substance P) produced additive stimulation of [³H]inositol phosphate production. Enhanced production of [³H]inositol phosphates was also observed under a variety of conditions known to cause smooth muscle depolarisation, including omission from the incubation medium of Na⁺ or K⁺, and in response to ouabain or veratridine.The results suggest that inositol lipid hydrolysis in visceral longitudinal smooth muscle may be triggered by depolarisation, an event which causes the entry of Ca²⁺ into the cell but which is not generally believed to cause the release of stored Ca²⁺ within the cell. However, calcium entry seems not to be essential for the effect on inositol lipid hydrolysis.     

Sertraline-induced desensitization of the serotonin 5HT-2 receptor transmembrane signaling system

Sertraline is a new, selective serotonin (5HT) uptake inhibitor with antidepressant activity. The effect of chronic administration of sertraline on 5HT-2 receptors in rat cortex was compared with that of the tricyclic antidepressant, amitriptyline. 5HT-2 receptors were evaluated in binding assays using [³H]-ketanserin and in functional assays of transmembrane signaling, hydrolysis of phosphoinositides. The daily injection of 17 mg/kg sertraline induced a desensitization of 5HT-2-mediated phosphoinositide hydrolysis after 28, but not 21, days. The administration of 1.2 mg/kg/day via continuous release pumps caused a more rapid desensitization. Amitriptyline administered chronically also produced a densensitization of the 5HT-2-mediated phosphoinositide hydrolysis response. A decrease in the density of 5HT-2 binding sites accompanies the functional desensitization after amitriptyline, but changes in 5HT-2 binding sites were not detected after chronic sertraline administration. Studies of the mechanism of action of sertraline show that the desensitization of the phosphoinositide hydrolysis response is homologous in nature, and that it is not secondary to changes in the synthesis of precursor lipids. Other possibilities such as alterations in coupling efficiency or in the activity of effector enzymes are currently being considered. The present results suggest a new postsynaptic action of antidepressant drugs at central 5HT-2 receptors (i.e., changes in 5HT-2 signal transduction at a site distal to the cell surface binding site) and illustrate the importance of studies of receptor signaling pathways to complement radioligand binding.     

Bradykinin-induced contraction of guinea pig lung in vitro

We have investigated the contractile effect of bradykinin (BK) in guinea pig lung in vitro. BK induces a dose-related contraction of lung parenchymal strips which is increased significantly in the presence of 10^–5 M captopril (an angiotensin converting enzyme inhibitor) or 10^–5 M dl-thiorphan (a neutral endopeptidase inhibitor). The kininase I inhibitor, dl-2-mercaptomethyl-3-guanidino-ethylthiopropionic acid (MGTPA), has no effect on the BK-induced contraction. BK is more potent in contracting parenchymal lung strips than other contractile agents (histamine, carbachol and substance P), however the BK-induced maximal contraction is lower than those obtained with histamine and carbachol. The B₁ agonist, des-Arg⁹-BK, does not contract lung parenchymal strips. The new BK B₂ receptor antagonists (Hoe 140, NPC 17731 and NPC 17761), which possess binding affinities in the nanomolar range, inhibit the BK-induced contractile response in a dose-dependent manner. The BK-induced contraction was unaffected by propranolol, atropine, tetrodotoxin, capsaicin pre-treatment, triprolidine, methysergide, Ro 19-3704 and N-nitro-l-arginine-methyl-ester (l-NAME), excluding the involvement of nervous pathways, preformed mast cell mediators, platelet-activating factor and nitric oxide. However, indomethacin, a cyclooxygenase inhibitor, AA-861, a 5-lipoxygenase inhibitor, and furegrelate, a thromboxane A₂ synthase inhibitor, decreased the contractile response to BK, suggesting that both cyclooxygenase and 5-lipoxygenase products are involved in this contraction. Thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ ATPase, abolished the BK-induced contraction demonstrating an intracellular calcium-dependent mechanism. Moreover, on a mixed lung cell suspension, obtained by enzymatic digestion, BK is able to induce phosphoinositide production. We conclude that BK, acting on B2 receptors, is a powerful contractile agent of the guinea pig lung in vitro. The BK-induced contraction, modulated by kininases II, is not dependent on neural mechanisms whereas both eicosanoids and intracellular calcium are involved. Correspondence to: J. P. Gies at the above address     

Altered membrane lipid composition in a human meningosarcoma

Summary In a sample of meningosarcoma, obtained at the time of surgery, the amount of total gangliosides and phospholipids was examined, together with the cholesterol content and the distribution of different ganglioside and phospholipid species. The phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4, 5-bisphosphate and phosphatidylcholine fatty acid composition was also analyzed. The ganglioside pattern in the meningosarcoma was different from the previously reported pattern in meningiomas of different histological origin, showing a higher concentration of GD₃, indicating that the so-called b pathway of ganglioside biosynthesis was the preferred one in this type of tumor; moreover the percentage content of polysialylated gangliosides was very low. Cholesterol and phospholipid content was lower than in meningiomas; the phosphatidylcholine increase and the sphingomyelin decrease would indicate a lower membrane microviscosity, a characteristic of tumor cells. Phosphoinositide and phosphatidylcholine fatty acid analysis revealed a considerable amount of docosahexaenoic acid. This abnormal presence of this fatty acid could lead to the production, after receptor stimulation, of a diacylglycerol containing docosahexaenoic acid, which, in turn, could be responsible for an altered activation pattern of protein kinase C, in this way promoting carcinogenesis.     

Localization of phospholipid-related signal molecules in salivary glands of rodents: A review

BackgroundIn the 1950s, Hokin conducted initial studies on phosphoinositide turnover/cycle in salivary glandular cells. From these studies, the idea emerged that receptor-mediated changes in intramembranous levels of phosphoinositides represent an early step in the stimulus-response pathway. Based on this idea and the general view that knowledge of the exact localization of a given endogenous molecule in cells in situ is important for understanding its functional significance, we have reviewed available information about the localization of several representative phosphoinositide-signaling molecules in the salivary glands in situ in mice.HighlightWe focused on phosphatidylinositol 4-kinase, phosphatidylinositol 4 phosphate 5-kinase α, β, γ, phospholipase Cβ, muscarinic cholinoceptors 1 and 3, diacylglycerol kinase ζ, phospholipase D1 and 2, ADP-ribosylation factor 6 and its exchange factors for Arf6, and cannabinoid receptors. These molecules individually exhibit differential localization in a spatiotemporal manner in the exocrine glands, making it possible to deduce their functional significance, such as their involvement in secretion and cell differentiation.ConclusionAlthough phosphoinositide-signaling molecules whose in situ localization in glandular cells has been clarified are still limited, the obtained information on their localization suggests that their functional significance is more valuable in glandular ducts than in acini. It thus suggests the necessity of greater attention to the ducts in their physio-pharmacological analyses. The purpose of this review is to encourage more in situ localization studies of phosphoinositide-signaling molecules with an aim to further understand their possible involvement in the pathogenesis of salivary gland diseases.