Hydrogen sulfide regulates cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells

2012 ◽  
Vol 302 (1) ◽  
pp. C59-C66 ◽  
Author(s):  
Ming Lu ◽  
Yi-Hong Liu ◽  
Chui Ying Ho ◽  
Chi Xin Tiong ◽  
Jin-Song Bian
Keyword(s):  
Hydrogen Sulfide ◽  
Adenylate Cyclase ◽  
Protein Level ◽  
Critical Role ◽  
Phosphodiesterase Inhibitor ◽  
Renin Activity ◽  
Renin Release ◽  
Intracellular Camp ◽  
Kidney Cells ◽  
Adrenoceptor Agonist

The present study aims to investigate the regulatory effect of hydrogen sulfide (H2S) on cAMP homeostasis and renin degranulation in As4.1 and rat renin-rich kidney cells. It was found in the present study that NaHS at 0.1–10 μM significantly decreased cAMP production in As4.1 cells treated with isoproterenol (a β-adrenoceptor agonist), forskolin (an adenylyl cyclase activator), or 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). NaHS at 10 μM suppressed adenylate cyclase activity but stimulated phosphodiesterase activity. We continued to study whether H2S may mediate cAMP-dependent renin degranulaion in As4.1 cells. It was found that NaHS at 0.1–10 μM significantly increased intracellular renin protein level. Moreover, NaHS reversed the declined renin content within As4.1 cells and normalized the upregulated renin activity in the culture medium of As4.1 cells treated with the above three stimuli. RT-PCR showed that cystathionine-γ-lyase is the main enzyme to produce endogenous H2S in As4.1 cells. Overexpression of cystathionine-γ-lyase increased endogenous H2S production and suppressed isoproterenol-induced renin release, suggesting that endogenous H2S may also inhibit renin release from As4.1 cells. We also tested whether H2S has a similar effect in renin-rich kidney cells. It was found that isoproterenol elevated intracellular cAMP level and extracellular renin activity but decreased renin protein level in the renin-rich kidney cells. Pretreatment with NaHS abolished these effects. In conclusion, H2S regulates cAMP homeostasis via inhibition of adenylate cyclase and stimulation of phosphodiesterase. Our findings suggest that H2S plays a critical role in regulation of renin degranulation in As4.1 and rat renin-rich kidney cells.

Alpha 2-adrenoceptor stimulation and cellular cAMP levels in microdissected rat glomeruli

1986 ◽  
Vol 250 (1) ◽  
pp. F103-F108
Author(s):  
S. Umemura ◽  
D. D. Smyth ◽  
W. A. Pettinger
Keyword(s):  
Adenylate Cyclase ◽  
Phosphodiesterase Inhibitor ◽  
Camp Accumulation ◽  
Camp Production ◽  
Camp Concentration ◽  
Adrenoceptor Agonist ◽  
Dose Dependent Fashion ◽  
Camp Levels ◽  
Dose Dependent ◽  
Camp Formation

A functional role for the numerically predominant glomerular alpha 2-adrenoceptors is unknown. In other tissues, activation of alpha 2-adrenoceptors inhibits adenylate cyclase activity. We therefore examined the effect of alpha 2-adrenoceptor stimulation with (-)-epinephrine (E) on the cellular cAMP concentration in glomeruli isolated by microdissection. Parathyroid hormone (1-34 PTH), prostaglandin E2 (PGE2), histamine, serotonin, or adenosine, in the presence of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and propranolol, was used to activate adenylate cyclase in single intact rat glomeruli. alpha 2-Adrenoceptors were activated with varying concentrations of E (37 degrees C, 2 min). In the presence of PTH-stimulated cAMP production, alpha 2-adrenoceptor activation with E (5 X 10(-7) to 5 X 10(-6) M) suppressed cellular cAMP levels in a dose-dependent fashion with the maximum at 30%. This suppression by E was inhibited by 5 X 10(-6) M yohimbine but not by 5 X 10(-6) M prazosin, confirming alpha 2-adrenoceptor mediation of this effect of E. Consistent with the above findings, the specific alpha 2-adrenoceptor agonist BHT933 inhibited PTH-stimulated cAMP accumulation. E also inhibited cAMP accumulation stimulated by serotonin. However, E did not suppress the PGE2-, histamine-, or adenosine-stimulated increase in cellular cAMP in the glomerulus. Activation of alpha 2-adrenoceptors inhibits cAMP formation stimulated by PTH or serotonin but not by PGE2, histamine, or adenosine in the rat glomerulus. Thus, the ability of alpha 2-adrenoceptors to inhibit adenylate cyclase appears to be hormone and probably function specific.

Interleukin-1 increases protein kinase A activity by a cAMP-independent mechanism in AtT-20 cells

1994 ◽  
Vol 266 (1) ◽  
pp. E79-E84 ◽  
Author(s):  
A. R. Gwosdow ◽  
N. A. O'Connell ◽  
A. B. Abou-Samra
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Protein Kinase A ◽  
Interleukin 1 ◽  
Phosphodiesterase Inhibitor ◽  
Intracellular Camp ◽  
Camp Accumulation ◽  
Acth Secretion ◽  
Intracellular Camp Accumulation ◽  
Kinase A

A recent study from this laboratory has shown that the inflammatory mediator, interleukin-1 alpha (IL-1 alpha), stimulates protein kinase A (PKA) activity and adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells without any detectable increase in intracellular cAMP accumulation. The present studies were conducted to determine if cAMP is involved in IL-1 alpha activation of PKA and if PKA is responsible for IL-1 alpha-induced ACTH release from AtT-20 cells. The data are consistent with a novel mechanism of PKA activation that does not involve cAMP. Inhibition of adenylate cyclase with 2'5'-dideoxyadenosine (2'5'-DDA) did not affect IL-1 alpha-induced increases in PKA activity and ACTH secretion. In contrast, CRF-stimulated PKA activity and ACTH secretion were inhibited by 2'5'-DDA. Additional evidence was obtained using the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX did not alter IL-1 alpha-induced PKA activity or ACTH secretion, yet IBMX potentiated CRF-induced cAMP accumulation. Inhibition of PKA with the PKA inhibitor, H-8, blocked activation of PKA and ACTH secretion by both IL-1 alpha and CRF in AtT-20 cells. These observations demonstrate that 1) the mechanism of IL-1 alpha activation of PKA is independent of adenylate cyclase or cAMP and 2) PKA is used by IL-1 alpha to induce ACTH secretion from AtT-20 cells.

scholarly journals Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells

2021 ◽  
Vol 22 (1) ◽  
pp. 397
Author(s):  
Nasir Javaid ◽  
Thuong L. H. Pham ◽  
Sangdun Choi
Keyword(s):  
Protein Level ◽  
Reference Genes ◽  
Mrna Level ◽  
High Specificity ◽  
Kidney Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Sox2 Expression

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

Enhancement of the Antihypertensive Effect of Hydrochlorothiazide in Dogs after Suppression of Renin Release by Beta-Adrenergic Blockade

1975 ◽  
Vol 48 (2) ◽  
pp. 147-151
Author(s):  
C. S. Sweet ◽  
M. Mandradjieff
Keyword(s):  
Blood Pressure ◽  
Plasma Renin Activity ◽  
Arterial Blood Pressure ◽  
Antihypertensive Effect ◽  
Plasma Renin ◽  
Renin Activity ◽  
Renin Release ◽  
Antihypertensive Activity ◽  
Adrenergic Blockade ◽  
Arterial Blood

1. Renal hypertensive dogs were treated with hydrochlorothiazide (8−2 μmol/kg or 33 μmol/kg daily for 7 days), or timolol (4.6 μmol/kg daily for 4 days), a potent β-adrenergic blocking agent, or combinations of these drugs). Changes in mean arterial blood pressure and plasma renin activity were measured over the treatment period. 2. Neither drug significantly lowered arterial blood pressure when administered alone. Plasma renin activity, which did not change during treatment with timolol, was substantially elevated during treatment with hydrochlorothiazide. 3. When timolol was administered concomitantly with hydrochlorothiazide, plasma renin activity was suppressed and blood pressure was significantly lowered. 4. These observations suggest that compensatory activation of the renin-angiotensin system limits the antihypertensive activity of hydrochlorothiazide in renal hypertensive dogs and suppression of diuretic-induced renin release by timolol unmasks the antihypertensive effect of the diuretic.

scholarly journals PGE2-mediated inhibition of T cell p59fynis independent of cAMP

1999 ◽  
Vol 277 (2) ◽  
pp. C302-C309 ◽  
Author(s):  
Mashkoor A. Choudhry ◽  
Zulfiqar Ahmed ◽  
Mohammed M. Sayeed
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adenylate Cyclase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Prostaglandin E ◽  
Intracellular Camp ◽  
Sprague Dawley ◽  
Cell Functions ◽  
Camp Levels

We recently observed that prostaglandin E2(PGE2)-mediated suppression of T cell functions could result from an attenuation of p59fynprotein tyrosine kinase activity. The present study evaluated the effects of an adenylate cyclase agonist (forskolin) and antagonist (SQ-22536), as well as those of cAMP analogues (dibutyryl cAMP and 8-bromo- cAMP), on T cell p59fynkinase activity. The study allowed us to assess whether PGE2-mediated activation of adenylate cyclase by itself or the elevation in intracellular cAMP levels is an integral event in the modulation of anti-CD3-linked p59fynactivation in T cells. The experiments were carried out with splenic T cells from male Sprague-Dawley rats. A 30–50% suppression in the autophosphorylation and the kinase activity of p59fynin T cells incubated with PGE2or forskolin was observed. Pretreatment of T cells with SQ-22536 prevented significant PGE2-mediated inhibition of T cell p59fynkinase activity. In contrast, no change in p59fynautophosphorylation and kinase activity in T cells treated with cAMP analogues was observed. These data suggest that PGE2-mediated suppression of p59fynautophosphorylation and kinase activity in T cells is dependent on the activation of adenylate cyclase and independent of the elevation in cAMP levels.

scholarly journals m6A Methyltransferase METTL14-Mediated Upregulation of Cytidine Deaminase Promoting Gemcitabine Resistance in Pancreatic Cancer

2021 ◽  
Vol 11 ◽  
Author(s):  
Congjun Zhang ◽  
Shuangyan Ou ◽  
Yuan Zhou ◽  
Pei Liu ◽  
Peiying Zhang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Western Blotting ◽  
Protein Level ◽  
Cytidine Deaminase ◽  
Critical Role ◽  
Human Pancreatic Cancer ◽  
Gemcitabine Resistance ◽  
Pancreatic Cancer Cells

ObjectivePancreatic cancer is one of the most lethal human malignancies. Gemcitabine is widely used to treat pancreatic cancer, and the resistance to chemotherapy is the major difficulty in treating the disease. N6-methyladenosine (m6A) modification, which regulates RNA splicing, stability, translocation, and translation, plays critical roles in cancer physiological and pathological processes. METTL14, an m6A Lmethyltransferase, was found deregulated in multiple cancer types. However, its role in gemcitabine resistance in pancreatic cancer remains elusive.MethodsThe mRNA and protein level of m6A modification associated genes were assessed by QRT-PCR and western blotting. Then, gemcitabine‐resistant pancreatic cancer cells were established. The growth of pancreatic cancer cells were analyzed using CCK8 assay and colony formation assay. METTL14 was depleted by using shRNA. The binding of p65 on METTL14 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Protein level of deoxycytidine kinase (DCK) and cytidine deaminase (CDA) was evaluated by western blotting. In vivo experiments were conducted to further confirm the critical role of METTL14 in gemcitabine resistance.ResultsWe found that gemcitabine treatment significantly increased the expression of m6A methyltransferase METTL14, and METTL14 was up-regulated in gemcitabine-resistance human pancreatic cancer cells. Suppression of METTL14 obviously increased the sensitivity of gemcitabine in resistant cells. Moreover, we identified that transcriptional factor p65 targeted the promoter region of METTL14 and up-regulated its expression, which then increased the expression of cytidine deaminase (CDA), an enzyme inactivates gemcitabine. Furthermore, in vivo experiment showed that depletion of METTL14 rescue the response of resistance cell to gemcitabine in a xenograft model.ConclusionOur study suggested that METTL14 is a potential target for chemotherapy resistance in pancreatic cancer.

scholarly journals Suppression of Platelet Aggregation byBordetella pertussis Adenylate Cyclase Toxin

1999 ◽  
Vol 67 (6) ◽  
pp. 2763-2768 ◽  
Author(s):  
Masaaki Iwaki ◽  
Kazunari Kamachi ◽  
Nikolaus Heveker ◽  
Toshifumi Konda
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Suppressive Effect ◽  
Intracellular Camp ◽  
Platelet Dysfunction ◽  
Camp Content ◽  
Point Mutant ◽  
Rabbit Platelets ◽  
Adenylate Cyclase Toxin ◽  
Induced Aggregation

ABSTRACT The effect of Bordetella pertussis adenylate cyclase toxin (ACT) on platelet aggregation was investigated. This cell-invasive adenylate cyclase completely suppressed ADP (10 μM)-induced aggregation of rabbit platelets at 3 μg/ml and strongly suppressed thrombin (0.2 U/ml)-induced aggregation at 10 μg/ml. The suppression was accompanied by marked increase in platelet intracellular cyclic AMP (cAMP) content and was diminished by the anti-ACT monoclonal antibody B7E11. A catalytically inactive point mutant of ACT did not show the suppressive effect. Since an increase of cAMP content is a known cause of platelet dysfunction, these results indicate that the observed platelet inactivation was due to the catalytic activity of ACT through increase of intracellular cAMP.

scholarly journals Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors

1997 ◽  
Vol 326 (2) ◽  
pp. 545-551 ◽  
Author(s):  
Edward S. TOBIAS ◽  
Enrique ROZENGURT ◽  
John M. C. CONNELL ◽  
Miles D. HOUSLAY
Keyword(s):  
Protein Kinase ◽  
Adenylate Cyclase ◽  
Protein Kinase D ◽  
Intracellular Camp ◽  
Camp Production ◽  
Camp Phosphodiesterase ◽  
Glucagon Receptor ◽  
Cos Cells ◽  
Protein Receptor ◽  
Dose Dependent Fashion

Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8±0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38–42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-α, PKC-βII and PKC-ϵ) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase.

Reversible effects of phenothiazines on frog gastric stimulation

1984 ◽  
Vol 247 (4) ◽  
pp. G366-G376
Author(s):  
N. Raphael ◽  
E. B. Ekblad ◽  
T. E. Machen
Keyword(s):  
Adenylate Cyclase ◽  
Time Course ◽  
Phosphodiesterase Inhibitor ◽  
Secretory Activity ◽  
Gastric Stimulation ◽  
Camp Content ◽  
Oxyntic Cell ◽  
Camp Generation ◽  
Stimulus Secretion Coupling

The calmodulin inhibitors trifluoperazine (TFP), chlorpromazine (CPZ), and promethazine (PZ) were tested for effects on stimulus-secretion coupling in in vitro bullfrog gastric mucosa. When added to histamine-stimulated tissues, the drugs caused H+ secretion to decrease and transepithelial resistance to increase over a 2-h time course. The potency sequence was TFP (IC50 = 40 microM) greater than CPZ (IC50 = 72 microM) congruent to PZ (IC50 = 72 microM). Anesthetics and other phenothiazines with weak anticalmodulin activity had no effect on secretory parameters. In the presence of histamine, further addition of isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) plus dibutyryl cAMP (DBcAMP), IBMX alone, or forskolin (a specific activator of adenylate cyclase) to phenothiazine-inhibited tissues caused full resumption of secretory activity. If TFP (50 microM) was added before stimulation with histamine, the normal increases in tissue cAMP content (which occurs primarily in oxyntic cells), oxyntic cell apical membrane elaboration (morphometric analysis of electron micrographs), and H+ secretion were all blocked. Subsequent addition of IBMX or IBMX plus DBcAMP completely reversed the TFP effect. These results indicate that the histamine-sensitive adenylate cyclase may be the site of TFP inhibition and Ca2+-calmodulin regulation; since these drugs inhibited stimulation by DBcAMP plus IBMX, they may also be exerting additional effects distal to cAMP generation.

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