cAMP induction by ouabain promotes endothelin-1 secretion via MAPK/ERK signaling in beating rabbit atria

Adenosine 3',5'-cyclic monophosphate (cAMP) participates in the regulation of numerous cellular functions, including the Na(+)-K(+)-ATPase (sodium pump). Ouabain, used in the treatment of several heart diseases, is known to increase cAMP levels but its effects on the atrium are not understood. The aim of the present study was to examine the effect of ouabain on the regulation of atrial cAMP production and its roles in atrial endothelin-1 (ET-1) secretion in isolated perfused beating rabbit atria. Our results showed that ouabain (3.0 micromol/L) significantly increased atrial dynamics and cAMP levels during recovery period. The ouabain-increased atrial dynamics was blocked by KB-R7943 (3.0 micromol/L), an inhibitor for reverse mode of Na(+)-Ca(2+) exchangers (NCX), but did not by L-type Ca2+ channel blocker nifedipine (1.0 micromol/L) or protein kinase A (PKA) selective inhibitor H-89 (3.0 micromol/L). Ouabain also enhanced atrial intracellular cAMP production in response to forskolin and theophyline (100.0 micromol/L), an inhibitor of phosphodiesterase, potentiated the ouabain-induced increase in cAMP. Ouabain and 8-Bromo-cAMP (0.5 micromol/L) markedly increased atrial ET-1 secretion, which was blocked by H-89 and by PD98059 (30 micromol/L), an inhibitor of extracellular-signal-regulated kinase (ERK) without changing ouabain-induced atrial dynamics. Our results demonstrated that ouabain increases atrial cAMP levels and promotes atrial ET-1 secretion via the mitogen-activated protein kinase (MAPK)/ERK signaling pathway. These findings may explain the development of cardiac hypertrophy in response to digitalis-like compounds.

Pituitary Adenylate Cyclase-activating Polypeptide Inhibits Pacemaker Activity of Colonic Interstitial Cells of Cajal

This study aimed to investigate the effect of pituitary adenylate cyclase-activating peptide (PACAP) on the pacemaker activity of interstitial cells of Cajal (ICC) in mouse colon and to identify the underlying mechanisms of PACAP action. Spontaneous pacemaker activity of colonic ICC and the effects of PACAP were studied using electrophysiological recordings. Exogenously applied PACAP induced hyperpolarization of the cell membrane and inhibited pacemaker frequency in a dose-dependent manner (from 0.1 nM to 100 nM). To investigate cyclic AMP (cAMP) involvement in the effects of PACAP on ICC, SQ-22536 (an inhibitor of adenylate cyclase) and cell-permeable 8-bromo-cAMP were used. SQ-22536 decreased the frequency of pacemaker potentials, and cell-permeable 8-bromo-cAMP increased the frequency of pacemaker potentials. The effects of SQ-22536 on pacemaker potential frequency and membrane hyperpolarization were rescued by co-treatment with glibenclamide (an ATP-sensitive K+ channel blocker). However, neither N(G)-nitro-L-arginine methyl ester (L-NAME, a competitive inhibitor of NO synthase) nor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ, an inhibitor of guanylate cyclase) had any effect on PACAP-induced activity. In conclusion, this study describes the effects of PACAP on ICC in the mouse colon. PACAP inhibited the pacemaker activity of ICC by acting through ATP-sensitive K+ channels. These results provide evidence of a physiological role for PACAP in regulating gastrointestinal (GI) motility through the modulation of ICC activity.

Effect of cAMP on short-circuit current in isolated human ciliary body / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cyclic adenosine monophosphate (cAMP) could activate chloride channels in bovine ciliary body and trigger an increase in the ionic current (short-circuit current, Isc) across the ciliary processes in pigs. The purpose of this study was to investigate how cAMP modulates Isc in isolated human ciliary processes and the possible involvement of chloride transport across the tissue in cAMP-induced Isc change.</p><p><b>METHODS</b>In an Ussing-type chamber system, the Isc changes induced by the cAMP analogue 8-bromo-cAMP and an adenylyl cyclase activator forskolin in isolated human ciliary processes were assessed. The involvement of Cl(-) component in the bath solution was investigated. The effect of Cl(-) channel (10 µmol/L niflumic acid and 1 mmol/L 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)), K(+) channel (10 mmol/L tetraethylammonium chloride (TEA)), or Na(+) channel blockers (1 mmol/L amiloride) on 8-bromo-cAMP-induced Isc change was also studied.</p><p><b>RESULTS</b>Dose-dependently, 8-bromo-cAMP (10 nmol/L-30 µmol/L) or forskolin (10 nmol/L-3 µmol/L) increased Isc across the ciliary processes with an increase in negative potential difference on the non-pigmented epithelium (NPE) side of the tissue. Isc increase induced by 8-bromo-cAMP was more pronounced when the drug was applied on the NPE side than on the pigmented epithelium side. When the tissue was bathed in low Cl(-) solutions, the Isc increase was significantly inhibited. Finally, niflumic acid and DIDS, but not TEA or amiloride, significantly prevented the Isc increase induced by 8-bromo-cAMP.</p><p><b>CONCLUSIONS</b>cAMP stimulates stroma-to-aqueous anionic transport in isolated human ciliary processes. Chloride is likely to be among the ions, the transportation of which across the tissue is triggered by cAMP, suggesting the potential role of cAMP in the process of aqueous humor formation in human eyes.</p>

Apoptosis of multiple myeloma cells induced by 8-Cl-cAMP in vitro / 中国实验血液学杂志

The study was aimed to investigate the possible effects of 8-chloroadenosine 3', 5'-monophosphate (8-Cl-cAMP) on the multiple myeloma cells. The multiple myeloma cell line RPMI8226 was used as in vitro models. The effect on growth inhibition of RPMI8226 cells was evaluated by cell growth and viability curve. DNA fragment was visualized by agarose gel electrophoresis. The amount of apoptosis cells was measured by flow cytometry. Meanwhile Western blot assay were used to detect the change of several key cell cycle regulatory proteins CDK2 and cyclin E in these cells before and after the treatment. The results showed that low dose 8-Cl-cAMP (1-30 micromol/L) inhibited the proliferation and viability of RPMI8226 cells significantly. Agarose gel electrophoresis of DNA revealed the apoptosis characteristic "ladder" pattern. Apoptosis was also confirmed by flow cytometry. In addition, 8-Cl-cAMP was able to inhibit the cell growth through modulating expression of cell cycle regulators CDK2 and cyclin E. It is concluded that 8-cl-cAMP inhibits the proliferation and induce apoptosis of multiple myeloma cells effectively.

Modulation of inflammatory cytokines by ATP-binding cassette A1 in THP-1 macrophages / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effects of ATP-binding cassette A1 (ABCA1) on intercellular cell adhesion molecule type 1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1) and interleukin-1beta (IL-1beta) in THP-1 macrophages stimulated with 8-Br-cAMP to identify a possible new mechanism that ABCA1 contributes to atherosclerogenesis (AS).</p><p><b>METHODS</b>Monocytic THP-1 cells were cultured in the presence of 100 nmol/L phorbol myristate acetate (PMA) for 72 h to transform the cells into THP-1 macrophages. After the macrophages were stimulated with 8-Br-cAMP (final concentration 0.5 mmol/L) for 3, 6, 12 and 24 h respectively, the amounts of ABCA1, ICAM-1 and MCP-1 mRNA were examined by real-time fluorescent quantitative RT-PCR, and the protein amounts of ABCA1, ICAM-1, MCP-1 and IL-1beta were determined by Western blotting and enzyme-linked immunosorbent assay (ELISA). Phosphorothioate antisense oligonucleotides of ABCA1 were add into the culture media at a final concentration of 100 nmol/L and the experiments were repeated.</p><p><b>RESULTS</b>ABCA1, ICAM-1 and MCP-1 mRNA and protein and IL-1beta protein were increased in the macrophages after stimulation with 8-Br-cAMP for 6 and 12 h. The mRNA expressions of ABCA1, ICAM-1 and MCP-1 were decreased significantly at 3 and 6 h (P<0.01), and the protein expressions of ABCA1, ICAM-1, MCP-1 and IL-1beta declined significantly at 12 and 24 h (P<0.01) after transfection of the macrophages with antisense oligonucleotides of ABCA1.</p><p><b>CONCLUSION</b>ABCA1 can increase the expressions of the inflammatory cytokines in THP-1 macrophages stimulated by 8-Br-cAMP and plays a role in the pathogenesis of AS.</p>

Effects of fenvalerate on steroidogenesis in cultured rat granulosa cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>This study was designed to examine the in vitro effects of fenvalerate on steroid production and steroidogenic enzymes mRNA expression level in rat granulosa cells.</p><p><b>METHODS</b>Using primary cultured rat granulosa cells (rGCs) as model, fenvalerate of various concentrations (0, 1, 5, 25, 125, 625 micromol/L) was added to the medium for 24 h. In some cases, optimal concentrations of 22(R)-hydroxycholesterol (25 micromol/L), Follicle stimulating hormone (FSH, 2 mg/L), or 8-Bromo-cAMP (1 mmol/L) were provided. Concentrations of 17 beta-estradiol(E2) and progesterone (P4) in the medium from the same culture wells were measured by RIA and the steroidogenic enzyme mRNA level was quantified by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>Fenvalerate decreased both P4 and E2 production in a dose-dependent manner while it could significantly stimulate rGCs proliferation. This inhibition was stronger in the presence of FSH. Furthermore, it could not be reversed by 22(R)-hydroxycholesterol or 8-Bromo-cAMP. RT-PCR revealed that fenvalerate had no significant effect on 3 beta-HSD, but could increase the P450scc mRNA level. In addition, 17 beta-HSD mRNA level was dramatically reduced with the increase of fenvalerate dose after 24 h treatment.</p><p><b>CONCLUSION</b>Fenvalerate inhibits both P4 and E2 production in rGCs. These results support the view that fenvalerate is considered as a kind of endocrine-disrupting chemicals. The mechanism of its disruption may involve the effects on steroidogenesis signaling cascades and/or steroidogenic enzyme's activity.</p>

Genome-wide expression profiling of 8-chloroadenosine- and 8-chloro-cAMP-treated human neuroblastoma cells using radioactive human cDNA microarray

Previous reports raised question as to whether 8-chloro-cyclic adenosine 3,5-monophosphate (8-Cl-cAMP) is a prodrug for its metabolite, 8-Cl-adenosine which exerts growth inhibition in a broad spectrum of cancer cells. The present study was carried out to clarify overall cellular affects of 8-Cl-cAMP and 8-Cl-adenosine on SK-N-DZ human neuroblastoma cells by ystematically characterizing gene expression using radioactive human cDNA microarray. Microarray was prepared with PCR-amplified cDNA of 2,304 known genes spotted on nylon membranes, employing (1)P-labeled cDNAs of SK-N-DZ cells as a probe. the expression levels of approximately 100 cDNAs, representing about 8% of the total DNA elements on the array, were altered in 8-Cl-adenosine- or 8-Cl-cAMP-treated cells, respectively. The genome-wide expression of the two samples exhibited partial overlaps; different sets of up-regulated genes but the same set of down-regulated genes. 8-Cl-adenosine treatment up- egulated genes involved in differentiation and development (LIM protein, connexin 26, neogenin, neurofilament triplet L protein and p21( WAF1/CIP1)) and immune response such as natural killer cells protein 4, and down-regulated ones involved in proliferation and transformation (transforming growth factor-beta, DYRK2, urokinase-type plasminogen activator and proteins involved in transcription and translation) which were in close parallel with those by 8-Cl-cAMP. Our results indicated that the two drugs shared common genomic pathways for the down-regulation of certain genes, but used distinct pathways for the up-regulation of different gene clusters. Based on the findings, we suggest that the anti-cancer activity of 8-Cl-cAMP results at least in part through 8-Cl-adenosine. Thus, the systematic use of DNA arrays can provide insight into the dynamic cellular pathways involved in anticancer activities of chemotherapeutics.

The effect of telomerase expression on the escape from M2 crisis in virus-transformed human retinal pigment epithelial cells

Integrins are heterodimeric glycoproteins that have been found to undergo dynamic temporal and spatial changes in the endometrium during the menstrual cycle and in early pregnancy. Specificity of integrins is known to be different in human endometrial stromal cells and decidual cells. These shifts of integrins suggested to play an important role in embryo implantation and can be modulated by progesterone, cAMP derivatives, and cytokines. The mechanisms of decidualization and its precise physiological role are still not clearly understood and in vitro systems could provide an alternative that overcomes limitations of studying such complex biological phenomena in vivo at the time of implantation. This study was undertaken to establish an in vitro model system for human decidualization using 8-bromo-cAMP and to investigate the characteristics of stromal integrin expression in vitro by 8-Br-cAMP. Endometrial stromal cells were isolated and cultured, and then were induced to decidualize by 0.5 mM 8-Br-cAMP for 15 days. Immunofluorescence staining and flow cytometric analyses of the integrin subunits (alpha1, alpha4, alpha5, alpha6, beta1 and alpha v beta3) were performed at day 9. In the presence of 8-Br-cAMP, the staining intensity of alpha v beta3 was significantly higher than control and measurements for alpha1, alpha4, alpha5, alpha6, and beta1 were similar. Immunofluorescent localization of the integrins reflected the differences obtained from the flow cytometric analyses described above. In summary, the expression of alpha;avbeta;b3 integrin increased in stromal cells in vitro decidualized by 8-Br-cAMP and this up-regulation of alpha v beta3 integrin expression during decidualization might influence on human implantation.

Protein kinase A-mediated phosphorylation of HERG potassium channels in a human cell line / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the molecular mechanism of human ether-a-go-go-related gene (HERG) potassium channels regulated by protein kinase A (PKA) in a human cell line.</p><p><b>METHODS</b>HERG channels were stably expressed in human embryonic kidney (HEK) 293 cells, and currents were measured with the patch clamp technique. The direct phosphorylation of HERG channel proteins expressed heterologously in Xenopus laevis oocytes was examined by (32)P labeling and immunoprecipitation with an anti-HERG antibody.</p><p><b>RESULTS</b>Elevation of the intracellular cAMP-concentration by incubation with the adenylate cyclase activator, forskolin (10 micromol/L), and the broad range phosphodiesterase inhibitor, IBMX (100 micromol/L), caused a HERG tail current reduction of 83.2%. In addition, direct application of the membrane permeable cAMP analog, 8-Br-cAMP (500 micromol/L), reduced the tail current amplitude by 29.3%. Intracellular application of the catalytic subunit of protein kinase A (200 U/ml) led to a tail current decrease by 56.9% and shifted the activation curve by 15.4 mV towards more positive potentials. HERG WT proteins showed two phosphorylated bands, an upper band with a molecular mass of approximately 155 kDa and a lower band with a molecular mass of approximately 135 kDa, indicating that both the core- and the fully glycosylated forms of the protein were phosphorylated.</p><p><b>CONCLUSIONS</b>PKA-mediated phosphorylation of HERG channels causes current reduction in a human cell line. The coupling between the repolarizing cardiac HERG potassium current and the protein kinase A system could contribute to arrhythmogenesis under pathophysiological conditions.</p>

The role of dependent pathway in eosinophil apoptosis / 천식및알레르기

BACKGROUND: Interleukin-5 (IL-5), IL-3, and GM-CSF are known to prolong the survival of eosinophils, and IL-5 has the most potent effect on eosinophil survivaL It is also known that divergent signals induce apoptosis in different cells. But, There have been few reports on about the intracellular signals that trigger the effectors of apoptosis. Cyclic AMP (cAMP) can modulate apoptosis in many cells. But, the role of intracellular cAMP in the IL-5 induced eosinophil survival is still not completely understood. OBJECTIVES: This study was aimed to elucidate the role of intracellular cAMP in IL-5 induced eosinophil survival. MATERIAL AND METHOD: Eosinophils were isolated from peripheral blood of atopic patients. Eosinophil viability was measured by means of propidium iodine (PI) method and the number of viable cells was counted by FAC scan (Becton Dickinson, USA). Cells were cultured with or without IL-5, and also with various cAMP-elevating agents (dibutyryl cAMP, 8-bromo-cAMP, N6- benzoyl cAMP). The concentrations of cAMP were measured by cAMP enzyme immunoassay system(BiotrakTM, Amersham). Finally cAMP dependent protein kinase A inhibitor (H8) was added to eosinophils to examine the effect of decreased intracellular cAMP activity on the viability of eosinophils stimulated with IL-5. RESULTS: The percentage of viable eosinophils was reduced rapidly from 92.1+/-1.8% to 8.23+/- 3.41% without IL-5 (p<0.05; n=ll, 4-day incubation). Upon addition of IL-5, it was increased to 33.02+7.8% (p<0.05; n=ll). In the absence of IL-5, the addition of cAMP-elevating agent increased eosinophil viability in a dose-dependent manner. Upon addition of H8 (24 uM), the eosinophil viability increased by IL-5 (52.5+/-6.4%) was significantly reduced to 27.2+/-5.4% (p<0.05;n=7). Compared with tissue culture media (TCM) only, IL-5 produced persistent elevation of intracellular cAMP of eosinophils in a time and dose dependent manner.

Effect of Vasoactive Intestinal Peptide on the Necrotic or Apoptotic Neuronal Death in Murine Cortical Cell Cultures

OBJECTIVE: The purpose of this study was to examine the effects of VIP on the apoptotic neuronal death induced by serum deprivation and the necrotic(excitotoxic) neuronal death by the exposure of NMDA in primary murine cortical cell cultures. MATERIALS AND METHODS: Near-pure neuronal cell cultures were prepared by plating fetal mice cortical cells onto polyethyleneimine-coated 24 well vessels. At 7 days in vitro(DIV), serum was removed from culture media for 24 hrs in near-pure neuronal cultures. Mixed cortical cell cultures containing both neurons and glia were prepared by plating fetal mice cortical cells onto an established monolayer of glia. At 12-14 DIV, excitotoxic neuronal death was induced by the addition of NMDA into the mixed cortical cultures. The neuronal cell death was assessed by either MTT or LDH assay after microscopic examination. RESULTS: Near-pure neuronal cell cultures deprived of serum undergo neuronal apoptosis marked by cell body shrinkage, chromatin condensation and DNA ladders. The apoptotic neuronal death following serum deprivation was significantly attenuated by the inclusion of a membrane-permeable cAMP analogue(8-bromo-cAMP; 100nM), an adenyl cyclase stimulator(forskolin; 10nM), a protein synthesis inhibitor(cycloheximide; 0.1ng/ml) or cell membrane depolarization(25mM KCl) during serum deprivation, but was not affected by the addition of an NMDA antagonist (MK-801; 10nM) or an antioxidant(trolox; 100nM). The inclusion of VIP(1, 3, 10nM) during deprivation also significantly prevented the neuronal death without dose-dependency. The neuroprotective effect of VIP(1nM) was not reversed by concomitant treatment with a VIP receptor antagonist([D-p-Cl-Phe6, Leu17]-VIP; 10, 30nM). Neuronglia co-cultures exposed to 300nM NMDA for 5 min produced neuronal death marked by cell body swelling. The neuronal death induced by the exposure of NMDA was markedly attenuated by MK-801 but not affected by 8-bromo-cAMP, forskolin, cycloheximide, high potassium or VIP. CONCLUSION: These results provide an evidence that VIP prevent apoptotic neuronal death induced by serum deprivation and suggest that VIP may have therapeutic potential for diseases in central nervous system linked to apoptotic neuronal death.

Agents that affect cAMP levels or protein kinase A activity modulate memory consolidation when injected into rat hippocampus but not amygdala

Male Wistar rats were trained in one-trial step-down inhibitory avoidance using a 0.4-mA footshock. At various times after training (0, 1.5, 3,6 and 9 h for the animals implanted into the CA1 region of the hippocampus; 0 and 3 h for those implanted into the amygdala), these animals received microinfusions of SKF38393 (7.5 mug/side), SCH23390 (0.5 mug/side), norepinephrine (0.3 mug/side), timolol (0.3 mug/side), 8-OH-DPAT (2.5 mug/side), NAN-190 (2.5 mug/side), forskolin (0.5 mug/side), KT5720 (0.5 mug/side) or 8-Br-cAMP (1.25 mug/side). Rats were tested for retention 24 h after training. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory whereas KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF38393, norepinephrine and NAN-190 caused memory facilitation, while KT5720, SCH23390, timolol and 8-OH-DPAT caused retrograde amnesia. Again, at 9 h after training, all treatments were inffective. When given into the amygdala, norepinephrine caused retrograde facilitation at 0 h after training. The other drugs infused into the amygdala did not cause any significant effect. These data suggest that in the hippocampus, but not in the amygdala, a cAMP/protein kinase A pathway is involved in memory cosolidation at 3 and 6 h after training, which is regulated by D1, Beta, and 5HT1A receptors. This correlates with data on increased post-training cAMP levels and a dual peak of protein kinase A activity and CREB-P levels (at 0 and 3-6 h) in rat hippocampus after training in this task. These results suggest that the hippocampus, but not the amygdala, is involved in long-term storage of step-down inhibitory avoidance in the rat.

Interaction of forskolin with the effect of N-6-cyclopentyladenosine on norepinephrine release in rat hippocampus

As it has been reported that the depolarization-induced norepinephrine (NE) release is modulated by activation of presynaptic A-1-adenosine heteroreceptor and various lines of evidence indicate the involvement of adenylate cyclase system in A-1-adenosine post-receptor mechanism in hippocampus, it was attempted to delineate the role of adenylate cyclase system in the A-1-receptor-mediated control of NE release in this study. Slices from rat hippocampus were equilibrated with (3H)-NE and the release of the labelled products was evoked by electrical stimulation (3 Hz, 5 V cm-1, 2 ms, rectangular pulses). The influence of various agents on the evoked tritium-outflow was investigated. N-6-Cyclopentyladenosine (CPA), a specific A-1-adenosine receptor agonist, in concentrations ranging from 0.1 to 10 micrometer decreased the (3H)-NE release in a dose-dependent manner without any change of basal rate of release. 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 micrometer), a selective A-1-receptor antagonist, inhibited the CPA effect. The responses to N-ethylmaleimide (3 & 10 micrometer), a SH-alkylating agent of G-protein, were characterized by increments of the evoked NE-release and the CPA effects were completely abolished by NEM pretreatment. Forskolin, a specific adenylate cyclase activator, in concentrations ranging from 0.1 to 30 micrometer increased the evoked and basal rate of NE release in a dose-dependent manner and the CPA effects were inhibited by forskolin pretreatment. Rolipram (1 & 10 micrometer), a phosphodiesterase inhibitor, did not affect the evoked NE release, but reduced the CPA effect. And 8-bromo-cAMP (100 & 300 micrometer), a membrane permeable cAMP analogue, inhibited the CPA effect significantly. These results suggest that the A-1-adenosine heteroreceptor plays an important role in NE-release via nucleotide-binding protein G-i in the rat hippocampus and that the adenylate cyclase system might be participated in this process.

Regulatory mechanisms of angiotensin II on the Na+/H+ antiport system in rabbit renal proximal tubule cells. I. Stimulatory effects of ANG II on Na+ uptake

The importance of the kidney in the development of hypertension was first demonstrated by Goldblatt and his colleagues more than fifty years ago. Many hormones and other regulatory factors have been proposed to play a major role in the development of hypertension. Among these factors angiotensin II (ANG II) is closely involved in renal hypertension development since it directly regulates Na+ reabsorption in the renal proximal tubule. Thus the aim of the present study was to examine signaling pathways of low dose of ANG II on the Na+ uptake of primary cultured rabbit renal proximal tubule cells (PTCs) in hormonally defined serum-free medium. The results were as follows: 1) 10-11 M ANG II has a significant stimulatory effect on growth as compared with control. Alkaline phosphatase exhibited significantly increased activity. However, leucine aminopeptidase and gamma-glutamyl transpeptidase activity were not significant as compared with control. In contrast to 10(-11) ANG II stimulated Na+ uptake (108.03 +/- 2.16% of that of control), 10(-9) M ANG II inhibited (92.42+/-2.23% of that of control). The stimulatory effect of ANG II on Na+ uptake was amiloride-sensitive and inhibited by losartan (ANG II receptor subtype 1 antagonist) and not by PD123319 (ANG II receptor subtype 2 antagonist). 2) Pertussis toxin (PTX) alone inhibited Na+ uptake by 85.52+/-3.52% of that of control. In addition, PTX pretreatment prevented the ANG II-induced stimulation of Na+ uptake. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), forskolin, and isobutylmethylxanthine (IBMX) alone inhibited Na+ uptake by 88.79+/-2.56, 80.63+/-4.38, and 84.47+/-4.74% of that of control, respectively, and prevented the ANG II-induced stimulation of Na+ uptake. However, 10(-11) M ANG II did not stimulate cAMP production. 3) The addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 0.01 ng/ml) to the PTCs produced significant increase in Na+ uptake (114.43+/-4.05% of that of control). When ANG II and TPA were added together to the PTCs, there was no additive effect on Na+ uptake. Staurosporine alone had no effect on Na+ uptake, but led to a complete inhibition of ANG II- or TPA-induced stimulation of Na+ uptake. ANG II treatment resulted in a 111.83 +/- 4.51% increase in total protein kinase C (PKC) activity. In conclusion, the PTX-sensitive PKC pathway is the main signaling cascade involved in the stimulatory effects of ANG II on Na+ uptake in the PTCs.

Effects of cyclic nucleotides and glipizide on the cardiovascular response of baclofen in the rats

The purpose of present study is to investigate the influence of a spinal gamma-aminobutyric acid B(GABA|B) receptor on a central regulation of blood pressure (BP) and heart rate (HR), and to define its mechanism in the spinal cord. In urethane-anesthetized, d-tubocurarine-paralyzed and artificially ventilated male Sprague-Dawley rats, intrathecal administration of drugs were carried out using injection cannula (33-gauge stainless steel) through the guide cannula (PE 10) which was inserted intrathecally at lower thoracic level through the puncture of a atlantooccipital membrane. Intrathecal injection of an GABA|B receptor agonist, baclofen (30, 60, 100 nmol) decreased both BP and HR dose-dependently. Pretreatment with 8-bromo-cAMP (50 nmol), a cAMP analog, or glipizide (50 nmol), a ATP-sensitive K+ channel blocker, attenuated the depressor and bradycardic effects of baclofen (100 nmol), but not with 8-bromo-cGMP (50 nmol), a cGMP analog. These results suggest that the GABA|B receptor in the spinal cord plays an inhibitory role in central cardiovascular regulation and that this depressor and bradycardic actions are mediated by the decrease of cAMP via the inhibition of adenylate cyclase and the opening of K+ channel.

Hormonal modulation of secretion of immunoreactive riboflavin carrier protein by adult rat Leydig cells in vitro.

Adult rat Leydig cells in culture synthesize and secrete riboflavin carrier protein (RCP) as demonstrated by [35S]-methionine incorporation into newly synthesized proteins followed by immunoprecipitation as well as specific radioimmunoassay. LH stimulates the secretion of RCP 4-fold which could be inhibited upto 75% by an aromatase inhibitor. 8-bromo-cyclic AMP and cholera toxin could mimic the LH stimulated secretion of the carrier protein. The extent of stimulation of RCP secretion brought about by exogenous estradiol-17 beta is comparable to that of LH. The antiestrogen tamoxifen, when added along with either LH or estrogen, inhibited the stimulated levels significantly. These results show that the estrogen-inducible riboflavin carrier is secreted by Leydig cells under positive regulation of LH.

Is asparagine-linked protein glycosylation an obligatory requirement for angiogenesis?

Dependence of protein N-glycosylation on capillary endothelial cell proliferation has been studied. Amphomycin, a potent N-glycosylation inhibitor, inhibited capillary endothelial cell proliferation in a dose-dependent manner. beta-Agonist isoproterenol as well as other intracellular cAMP enhancing agents, viz. cholera toxin, prostaglandin E1 and 8Br-cAMP, also enhanced capillary endothelial cell proliferation. In addition to cell proliferation, isoproterenol also enhanced protein glycosylation in these cells. Isoproterenol effect was mediated by beta-adrenoreceptors, as it got reduced on pre-treatment of cells with either atenolol or ICI 118, 551 or propranolol. Furthermore, isoproterenol stimulation of protein glycosylation by exogenous dolichyl monophosphate and its inhibition by tunicamycin (GlcNAc-1P transferase inhibitor) supported the concept that isoproterenol specifically stimulated protein N-glycosylation event(s) in the cell.