Milrinone is a positive inotropic/vasodilator agent in both preclinical and clinical studies. Milrinone has been shown previously to inhibit specific type III PDE isolated from different sources. To investigate the effect and the mechanism of milrinone on the corpus cavernosum, we have studied on the human and rabbit corpus cavernosum using organ bath and measured the levels of cAMP and cGMP after treatment of milrinone and papaverine. Our results show that milrinone relaxes human and rabbit corpus cavernosal tissue in a dose- dependent manner. And significant increases in cAMP content were evident with milrinone. These indicate that the accumulation of cAMP resulting from the inhibition of type III PDE plays an important role in milrinone on rabbit corpus cavernosum. And these may reflect the impotence of cAMP dependent second messenger systems for the relaxation of penile smooth muscle. These results suggest a possible potential for milrinone in the treatment of impotence.
Baths ; Erectile Dysfunction ; Humans* ; Male ; Milrinone* ; Muscle, Smooth ; Papaverine ; Relaxation ; Second Messenger Systems

Phospholipase C (PLC)1 hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 induces a transient increase in intracellular free Ca2+, while DAG directly activates protein kinase C. Upon stimulation of cells with growth factors, PLC-gamma1 is activated upon their association with and phosphorylation by receptor and non-receptor tyrosine kinases. In this review, we will focus on the activation mechanism and regulatory function of PLC-gamma1.
Cell Division ; Enzyme Activation ; Isoenzymes/metabolism* ; Phospholipase C/metabolism* ; Second Messenger Systems ; T-Lymphocytes

BACKGROUND: Intracellular ionized calcium plays a central role in the transduction of external stimuli as a critical second messenger. The spectral properties of fluo-3 allows the analysis of intracellular ionized calcium level by flow cytometers. The aim of this study is to assess the performance of flow cytometer for measuring intracellular ionized calcium level using fluo-3 and to define the reference interval of intracellular ionized calcium level of lymphocytes from healthy people, and to find out the clinical implications according to various disorders. METHODS: For the analytical performance of flow cytometer on determining the concentration of intracellular ionized calcium, precision study, lowest limit of detection, analytical range, and the loading stability of fluo-3 were per foamed. Fifty-four cases of healthy people, 52 cases of renal transplant patients, and 20 cases of diabetes mellitus patients were included in this study. RESULTS: Loading effect of fluo-3 at room temperature was stable upto 5 hours. Lowest limit of detection of ionized calcium concentration was 4.34 nM at in-situ calibration procedure. Within-run and among-day intraindividual CVs of in-situ calibration procedure were 6.67% and 13.99% respectively, and of optical calibration procedure were 13.86% and 16.12% respectively. The reference interval of cytosolic free calcium level for healthy people ranged 73.54 - 155.09 nM without sexual differences. The level of intracellular ionized calcium was lowered by 36.9% on renal transplant group in comparison with healthy control group. But, level of cytosolic free calcium was Increased upto 276.0% on acute rejection group and 159.1% on diabetes mellitus group compared to control group. CONCLUSIONS: These results reveal that in-situ calibration method for intra cellular ionized calcium using flow cytometry with flue-3 can be regarded as an accurate and standardized method. Quantitation of intracellular ionized calcium level might be used as the monitoring test for early detection of acute rejection after renal transplantation.
Calcium* ; Calibration ; Cytosol ; Diabetes Mellitus ; Flow Cytometry* ; Humans* ; Kidney Transplantation ; Limit of Detection ; Lymphocytes* ; Second Messenger Systems

Objective: To establish a detection method based on Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) that can sensitively detect the second messenger cyclic AMP (cAMP) in the cytoplasm. Methods: The eukaryotic expression vectors of CFTR and YFP-H148Q / I152L were constructed respectively. FRT cells co-expressing CFTR and YFP-H148Q / I152L were obtained by liposome transfection. The expression of CFTR and YFP-H148Q / I152L in FRT cells was observed by an inverted fluorescence microscopy, and flow cytometry was used to detect the purity of cells; The cell model was identified by the fluorescence quenching kinetics test. The validation of the cell model which could screen CFTR modulators was verified by the fluorescence quenching kinetics experiments. The radioimmunoassay was used to detect the cAMP concentration in cytoplasm after adding CFTR activator. Results: The results of the inverted fluorescence microscope showed that CFTR was expressed in the cell membrane and YFP-H148Q / I152L was expressed in the cytoplasm of FRT cells. The FRT cell model stably co-expressing ANO1 and YFP-H148Q / I152L was successfully constructed. The model could screen CFTR modulators, and the slope of fluorescence change and the concentration of CFTR modulators were in a dose-dependent manner. The slope of the fluorescence could reflect the cAMP concentration in the cytoplasm. The cell model could sensitively detect the intracellular cAMP concentration. Conclusion: The cell model could efficiently and sensitively detect the second messenger cAMP concentration in the cytoplasm, and it provided a simple and efficient method for the study of other targets associated cAMP signal.
Cyclic AMP ; Cystic Fibrosis Transmembrane Conductance Regulator ; Cytoplasm ; Second Messenger Systems

Understanding the cellular and molecular mechanisms involved in the development and progression of pulmonary hypertension (PH) remains imperative if we are to successfully improve the quality of life and life span of patients with the disease. A whole plethora of mechanisms are associated with the development and progression of PH. Such complexity makes it difficult to isolate one particular pathway to target clinically. Changes in intracellular free calcium concentration, the most common intracellular second messenger, can have significant impact in defining the pathogenic mechanisms leading to its development and persistence. Signaling pathways leading to the elevation of [Ca2+]cyt contribute to pulmonary vasoconstriction, excessive proliferation of smooth muscle cells and ultimately pulmonary vascular remodeling. This current review serves to summarize the some of the most recent advances in the regulation of calcium during pulmonary hypertension.
Calcium ; Humans ; Hydrogen-Ion Concentration ; Hypertension, Pulmonary ; Myocytes, Smooth Muscle ; Quality of Life ; Second Messenger Systems ; Vasoconstriction

Using murine immortalized microglial cells (BV-2), we examined the regulation of RANTES production stimulated by lipopolysaccharide (LPS), focusing on the role of mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-kappaB. The result showed that RANTES (regulated upon activation of normal T cell expressed and secreted) was induced at the mRNA and protein levels in a dose- and time-dependent manner in response to LPS. From investigations of second messenger pathways involved in regulating the secretion of RANTES, we found that LPS induced phosphorylation of extracellular signal-regulated kinase (Erk), p38 MAPK and c-Jun-N-terminal kinase (JNK), and activated NF-kappaB. To determine whether this MAPK phosphorylation is involved in LPS-stimulated RANTES production, we used specific inhibitors for p38 MAPK and Erk, SB 203580 and PD 98059, respectively. LPS-induced RANTES production was reduced approximately 80% at 25 micrometer of SB 203580 treatment. But PD 98059 did not affect RANTES production. Pyrrolidine-dithiocarbamate (PDTC), NF-kappaB inhibitor, reduced RANTES secretion. These results suggest that LPS-induced RANTES production in microglial cells (BV-2) is mainly mediated by the coordination of p38 MAPK and NF-kappaB cascade.
Chemokine CCL5* ; Microglia* ; NF-kappa B* ; p38 Mitogen-Activated Protein Kinases* ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; RNA, Messenger ; Second Messenger Systems

Sphingosine-1-phosphate (S1P) is emerging as a new class of second messenger involved in cellular proliferation, differentiation, and apoptosis and is implicated in diverse physiological functions. Despite many studies on the biological functions of S1P, however, little is known about its role in neuronal differentiation. By use of reverse transcription-polymerase chain reaction and immunostaining, this study aimed to explore whether S1P can differentiate neuroblastoma cells into neural cells. After incubation with 1 uM or 10 uM S1P, the number of neurite-bearing cells increased. Furthermore, the neuroblastoma cells revealed immunoreactivity for neural-specific markers such as GAP43, NFH, and SYP by immunostaining. The expression of NFH, MAP2, SYP, NeuroD1, and SYT mRNA, which is specific for neurons, was increased as shown by RT-PCR studies. The results of this study suggest that that S1P can induce neuronal differentiation and may be a good candidate for the treatment of neurodegenerative diseases.
Apoptosis ; Cell Differentiation ; Cell Proliferation ; Lysophospholipids ; Neurites ; Neuroblastoma ; Neurodegenerative Diseases ; Neurons ; RNA, Messenger ; Second Messenger Systems ; Sphingosine

Estrogen - the female sexual hormone playing the most important role - plays a physiologically significant role, not only regulating in cell signals with second messenger but also being active in regulating transcription. Estrogen receptor (ER) which is a protein accepting estrogen not only play the role of a transcription factor combining with other genes to regulate their activity like other nuclear receptors but also performs external activities, combining with DNA, etc. G-protein coupled ER (GPER) that has been recently discovered exists as 7-membrane and has non-genomic (rapid) signaling. These functions, however, are not extensively addressed. This paper discusses the roles of GPER and its physiological mechanism.
DNA ; Estradiol ; Estrogens* ; Female ; Genomics ; GTP-Binding Proteins* ; Humans ; Receptors, Cytoplasmic and Nuclear ; Second Messenger Systems ; Transcription Factors ; Women's Health*

BACKGROUND: The potent bronchodilatory effects of ketamine on airway smooth muscle tone are important in the management of patients with asthma, but its mode of action is unclear. In the present study we evaluated that effects on isolated guinea pig tracheal smooth muscle. METHODS: Changes of isometric contraction of strip were measured. (1) Serial stimulation with acetylcholine(ACh) in Krebs solution or with A23187, nifedipine, ketamine were evaluated. After that, ACh stimulation was induced in Ca2+ free solution. (2) In Ca2+ free solution, ACh contraction was obtained(L1) and emptied by repetitive ACh stimulation. Internal stores were refilled by Ca2+ with ACh stimulation. During the incubation period, A23187, nifedipine, ketamine, cyclopiazonic acid + ketamine was added and tested for their ability to inhibit refilling. Refilling was evaluated by ACh produced contraction (L2) with ratio (L2/L1). (3) Effects of ketamine on the contraction induced by caffeine were also checked. RESULTS: Ketamine inhibited amplitude dose-dependently by successive application of ACh in modified Krebs solution and Ca2+ free solution. Ca2+ influx through voltage gated channels were inhibited with nifedipine but not with A23187. ACh sensitive internal store were different when A23187, nifedipine and ketamine were applied in Ca2+ free solution. Refilling of internal store were potentiated by A23187, but decreased by nifedipine and ketamine. Caffeine produced contractions in the presence of ketamine were not significantly different from control. CONCLUSION: We concluded that the inhibitory effects of ketamine in guinea pig trachea were by acting through voltage and receptor gated channels in dose-depedent manner and these effects may be interferences of intracellular second messengers system.
Animals ; Asthma ; Caffeine ; Calcimycin ; Guinea Pigs* ; Guinea* ; Humans ; Isometric Contraction ; Ketamine* ; Muscle, Smooth ; Nifedipine ; Second Messenger Systems ; Trachea*

BACKGROUND AND OBJECTIVES: Airway mucus hypersecretion is one of the major clinical manifestation of patients suffering from various upper or lower respiratory tract diseases. But unfortunately, no drugs are yet available for controlling the airway mucus hypersecretion. Although pharmacological approaches for controlling airway mucus production is currently limited, among the few useful drugs, steroid hormones seem to be the most effective. The effect of steroid hormones is classically described as that of a genomic mechanism involving nucleus transcription. but there is a growing evidence for rapid, non-genomic effect of steroid hormones working through various second messenger systems and ion transporters. MATERIALS AND METHOD: In order to investigate a possible rapid, non-genomic effect of dexamethasone and aldosterone in cultured normal human nasal epithelial (NHNE) cell, the effect of dexamethasone and aldosterone was tested for intracellular calcium response (delta[Ca2+]i) and mucin secretion to external ATP, a known secretagogue in airway epithelial cells, with fluorescence imaging system and ELISA, respectively. Also, we demonstrated the effect of dexamethasone on the mRNA expression of MUC5AC mucin gene for evidence of the existence of non-genomic mechanism of steroid hormones in NHNE cells. RESULTS: Pretreatment with dexamethasone or aldosterone with various concentrations and duration caused a reduction in the delta[Ca2+]i and mucin secretion to ATP. In RT-PCR, Ten minutes dexamethasone pretreatment did not attenuate the mRNA expression of MUC5AC mucin gene, but 24 hours of dexamethasone pretreatment did so. These data indicate that a few minutes of steroid hormone pretreatment can decrease the delta[Ca2+]i and mucin secretion via a non-genomic mechanism. CONCLUSION: In this study, we confirmed the rapid, non-genomic effect of steroid hormones in NHNE cells and suggest this study as a research model for developing antisecretory drugs that have rapid effect.
Adenosine Triphosphate ; Aldosterone ; Calcium ; Dexamethasone ; Enzyme-Linked Immunosorbent Assay ; Epithelial Cells* ; Humans* ; Ion Transport ; Mucins* ; Mucus ; Nasal Mucosa ; Optical Imaging ; Respiratory Tract Diseases ; RNA, Messenger ; Second Messenger Systems