Biomedical subjects

D P Brooks

Publications and source records attributed to D P Brooks.

At least 19 recordsLinked to original sources

PubMed2005

Female, but not male, serotonin reuptake transporter (5-HTT) knockout mice exhibit bladder instability.

Correlations exist between the incidence of depression, irritable bowel syndrome (IBS) and overactive bladder [Masand, P.S., Kaplan, D.S., Gupta, S., Bhandary, A.N., Nasra, G.S., Kline, M.D., Margo, K.L., 1995. Major depression and irritable bowel syndrome: is there a relationship? J. Clin. Psychiatry 56, 363-367.; Cukier, J.M., Cortina-Borja, M., Brading, A.F., 1997. A case-control study to examine any association between idiopathic detrusor instability and gastrointestinal tract disorder, and between irritable bowel syndrome and urinary tract disorder. Br. J. Urol. 79, 865-878.; Monga, A.K., Marrero, J.M., Stanton, S.L., Lemieux, M.C., Maxwell, J.D., 1997. Is there an irritable bladder in the irritable bowel syndrome? Br. J. Obstet. Gynaecol. 104, 1409-1412.; Zorn, B.H., Montgomery, H., Pieper, K., Gray, M., Steers, W.D., 1999. Urinary incontinence and depression. J. Urol. 162, 82-84.]. Furthermore, alterations in serotonergic neurotransmission may play a common role in the etiology of these disorders. Serotonin reuptake transporter knockout mice (5-HTT(-/-)) display phenotypes consistent with clinical features of mood and bowel disorders including anxiety and abnormal gastrointestinal motility [Holmes, A., Murphy, D.L., Crawley, J.N., 2003. Abnormal behavioral phenotypes of serotonin transporter knockout mice: parallels with human anxiety and depression. Biol. Psychiatry 54, 953-959.]. In the present study, we evaluated bladder function in 5-HTT(-/-) mice. We have found that female 5-HTT(-/-) mice exhibit bladder dysfunction, characterized by significant increases in the frequency of spontaneous non-voiding bladder contractions and decreases in void volume compared to control female mice. These differences were not observed in male knockout mice. These studies provide significant supporting data for a mechanistic link between alterations in 5-HT, depression, IBS and overactive bladder in women.

Animals↗

PubMed2001

Role of caspases in human renal proximal tubular epithelial cell apoptosis.

In the present study, we have used an in vitro model of apoptosis using primary human renal proximal tubular epithelial (RPTE) cells to investigate the mechanisms involved in renal cell apoptosis. Treatment of RPTE cells with okadaic acid for 24-48 h induced apoptosis in a concentration-dependent manner. Apoptosis was accompanied by the activation of the p38 mitogen-activated protein kinase (MAPK) pathway followed by the activation of caspase-9, -3, and -7. The induction of caspase activity correlated with the proteolytic cleavage of beta-catenin, suggesting that beta-catenin is a caspase substrate. The caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), resulted in a dose-dependent inhibition of apoptosis and beta-catenin cleavage. These data suggest that okadaic acid-induced apoptosis is p38 MAPK and caspase-dependent and that proteolytic cleavage of beta-catenin by caspases is likely to be a downstream molecular event associated with the morphological and cytoskeletal changes induced during apoptosis.

Amino Acid Chloromethyl Ketones↗

PubMed2001

Hypertensive end-organ damage and premature mortality are p38 mitogen-activated protein kinase-dependent in a rat model of cardiac hypertrophy and dysfunction.

BACKGROUND: Numerous pathological mediators of cardiac hypertrophy (eg, neurohormones, cytokines, and stretch) have been shown to activate p38 MAPK. The purpose of the present study was to examine p38 MAPK activation and the effects of its long-term inhibition in a model of hypertensive cardiac hypertrophy/dysfunction and end-organ damage. METHODS AND RESULTS: In spontaneously hypertensive stroke-prone (SP) rats receiving a high-salt/high-fat diet (SFD), myocardial p38 MAPK was activated persistently during the development of cardiac hypertrophy and inactivated during decompensation. Long-term oral treatment of SFD-SP rats with a selective p38 MAPK inhibitor (SB239063) significantly enhanced survival over an 18-week period compared with the untreated group (100% versus 50%). Periodic echocardiographic analysis revealed a significant reduction in LV hypertrophy and dysfunction in the SB239063-treatment groups. Little or no difference in blood pressure was noted in the treatment or vehicle groups. Basal and stimulated (lipopolysaccharide) plasma tumor necrosis factor-alpha concentrations were reduced in the SB239063-treatment groups. In vitro vasoreactivity studies demonstrated a significant preservation of endothelium-dependent relaxation in animals treated with the p38 MAPK inhibitor without effects on contraction or NO-mediated vasorelaxation. Proteinuria and the incidence of stroke (53% versus 7%) were also reduced significantly in the SB239063-treated groups. CONCLUSIONS: These results demonstrate a crucial role for p38 MAPK in hypertensive cardiac hypertrophy and end-organ damage. Interrupting its function with a specific p38 MAPK inhibitor halts clinical deterioration.

Animals↗

PubMed2001

The role of transforming growth factor-beta and its receptors in human prostate smooth muscle cell fibronectin production.

In the present study, the role of transforming growth factor-beta (TGFbeta) on the production of the extracellular matrix component, fibronectin, in the prostate has been studied. The mRNA levels of fibronectin, TGFbeta and the two TGFbeta receptors, ALK5 (activin like kinase) and type II, were measured using reverse-transcription polymerase chain reaction (RT-PCR). TGFbeta increased fibronectin mRNA and protein (7-fold) in a concentration-dependent fashion. An interesting relationship between the two TGFbeta receptors was found in that TGFbeta caused an upregulation of its type I receptor mRNA (5-6-fold) and a downregulation of the type II receptor mRNA (5-fold). Time-course experiments revealed that the change in expression of the TGFbeta receptors reached maximum at 24 h with an early increase at 4-5 h, whereas the fibronectin gene expression was not significantly stimulated until about 24 h. These data provide evidence that TGFbeta stimulates extracellular matrix production in prostate cells.

Activin Receptors, Type I↗

PubMed2001

Eprosartan reduces cardiac hypertrophy, protects heart and kidney, and prevents early mortality in severely hypertensive stroke-prone rats.

OBJECTIVE: Eprosartan is a selective angiotensin II type I receptor antagonist approved for the treatment of hypertension. In the present studies, eprosartan's ability to provide end-organ protection was evaluated in a model of cardiomyopathy and renal failure in stroke-prone rats (SP). METHODS: SP were fed a high fat (24.5% in food) and high salt (1% in water) diet (SFD). Eprosartan (60 mg/kg/day) or vehicle (saline control) (n = 25/group) was administered by intraperitoneally-implanted minipumps to these SP on the SFD for 12 weeks. Normal diet fed SP and WKY rats (n = 25/group) were also included for comparison (i.e. served as normal controls). Mortality, hemodynamics, and both renal and cardiac function and histopathology were monitored in all treatment groups. RESULTS: Eprosartan decreased the severely elevated arterial pressure (-12%; P < 0.05) produced by SFD but did not affect heart rate. Vehicle-treated SP-SFD control rats exhibited significant weight loss (-13%; P < 0.05) and marked mortality (50% by week 6 and 95% by week 9; P < 0.01). Eprosartan-treated SP-SFD rats maintained normal weight, and exhibited zero mortality at week 12 and beyond. Eprosartan prevented the increased urinary protein excretion (P < 0.05) that was observed in vehicle-treated SP-SFD rats. Echocardiographic (i.e. 2-D guided M-mode) evaluation indicated that SP-SFD vehicle control rats exhibited increased septal (+22.2%) and posterior left ventricular wall (+30.0%) thickness, and decreased left ventricular chamber diameter (-15.9%), chamber volume (-32.7%), stroke volume (-48.7%) and ejection fraction (-22.3%), and a remarkable decrease in cardiac output (-59.3%) compared to controls (all P < 0.05). These same parameters in eprosartan-treated SP-SFD rats were normal and differed markedly and consistently from vehicle-treated SP-SFD rats (i.e. treatment prevented pathology; all P < 0.05). Cardiac-gated MRI data confirmed the ability of eprosartan to prevent cardiac pathology/remodeling (P < 0.05). Histopathological analysis of hearts and kidneys indicated that eprosartan treatment significantly reduced end-organ damage (P < 0.01) and provided corroborative evidence that eprosartan reduced remodeling of these organs. Vehicle-treated SP-SFD rats exhibited a 40% increase in the plasma level of pro-atrial natiuretic factor that was reduced to normal by eprosartan (P < 0.05). CONCLUSION: These data demonstrate that eprosartan, at a clinically relevant dose, provides significant end-organ protection in the severely hypertensive stroke-prone rat. It preserves cardiac and renal structural integrity, reduces cardiac hypertrophy and indices of heart failure, maintains normal function of the heart and kidneys, and eliminates premature mortality due to hypertension-induced end-organ failure.

Acrylates↗

PubMed2001

Renoprotective effects of carvedilol in hypertensive-stroke prone rats may involve inhibition of TGF beta expression.

1. The effect of carvedilol on renal function, structure and expression of TGF beta and the matrix proteins fibronectin, collagen I and collagen III, was evaluated in spontaneously hypertensive stroke-prone (SHR-SP) rats fed a high fat, high salt diet. 2. Carvedilol treatment for 11 to 18 weeks did not alter systolic blood pressure in SHR-SP rats, however, it resulted in a significant reduction in heart rate. 3. Carvedilol treatment reduced renal fibrosis and total, active and chronic renal damage to levels approaching those of WKY rats on a normal diet. 4. Urinary protein excretion was higher in SHR-SP rats (51+/-10 mg day(-1)) than WKY rats (18+/-2 mg day(-1)) and this was further increased when SHR-SP rats were fed a high fat, high salt diet (251+/-120 mg day(-1)). Treatment with carvedilol resulted in significantly lower urinary protein excretion (37+/-15 mg day(-1)). 5. The expression of TGF beta mRNA was significantly higher in SHR-SP rats compared to WKY rats and a further increase was observed when rats were fed a high fat, high salt diet. Renal TGF beta expression was significantly reduced by treatment with carvedilol. The expression of fibronectin and collagen I and collagen III mRNA showed a pattern similar to that observed with TGF beta mRNA expression. Collagen I mRNA expression followed a pattern similar to renal fibrosis. 6. These data indicate that carvedilol can provide significant renal protection in the absence of any antihypertensive activity and that the mechanisms involved in this action may include reduced expression of profibrotic factors such as TGF beta.

Adrenergic beta-Antagonists↗

PubMed2001

Parathyroid hormone-1 receptor down-regulation in kidneys from rats with chronic renal failure.

Although secondary hyperparathyroidism is a common complication of chronic renal failure, few studies have examined the characteristics of parathyroid hormone (PTH) binding to the kidney or the regulation of the PTH receptor in chronic renal disease. In this study we measured PTH binding to the PTH-1 receptor in renal cortical membranes from normal rats and from rats with experimentally induced chronic renal failure. In normal rats, analysis of saturation binding experiments using 125I PTH-related peptide (chicken, cPTHrP) revealed apparent Kd and Bmax values of 1.16 +/- 0.14 nmol/l and 338 +/- 22.7 fmol/mg, respectively. Three weeks following induction of renal failure there was no change in the affinity of the PTH-1 receptor (Kd = 1.51 +/- 0.24 nmol/l) but the Bmax was reduced by 45% (183 +/- 32.5). In normal rats which had undergone thyroparathyroidectomy, the Kd was unchanged (1.17 +/- 0.09) while the Bmax increased to 459 +/- 31 fmol/mg. We conclude that chronic renal failure is accompanied by a downregulation of renal PTH-1 receptors.

Animals↗

PubMed2001

Dopamine inhibits vasopressin action in the rat inner medullary collecting duct via alpha(2)-adrenoceptors.

We compared the effects of dopamine and norepinephrine on vasopressin (AVP)-stimulated increases in osmotic water permeability (Pf) and cAMP accumulation in the rat inner medullary collecting duct (IMCD). Both dopamine and norepinephrine inhibited AVP-induced Pf and cAMP accumulation in a concentration-dependent manner; however, norepinephrine was approximately 100-fold more potent than dopamine. The effects of dopamine on Pf were antagonized by the selective alpha(2)-adrenoceptor antagonist, rauwolscine (10 nM--1 microM). Clozapine (10 microM), a dopamine D(4) receptor antagonist with significant activity at adrenergic receptors, partially attenuated both dopamine and norepinephrine-induced decreases in AVP-stimulated Pf. Dopamine-induced inhibition of AVP-dependent cAMP levels was antagonized by the alpha(2)-adrenoceptor antagonists, rauwolscine, idazoxan, and yohimbine, but not by the dopamine receptor antagonists, spiperone, SCH-23390, or raclopride. Clozapine (1--10 microM) inhibited the effects of both dopamine and norepinephrine on AVP-stimulated cAMP levels. We conclude that the inhibitory effects of dopamine on AVP-induced Pf and cAMP accumulation in the rat IMCD are mediated via alpha(2)-adrenoceptors.

Adrenergic alpha-Antagonists↗

PubMed2000

Gene expression in rats with renal disease treated with the angiotensin II receptor antagonist, eprosartan.

The role of ANG II on renal and cardiac gene expression of matrix proteins was studied in rats with progressive renal disease. Induction of renal failure by five-sixths nephrectomy of Sprague-Dawley rats resulted in hypertension (163 +/- 19 vs. control pressures of 108 +/- 6 mmHg), proteinuria (83 +/- 47 vs. 14 +/- 2 mg/day), and increased renal expression of fibronectin, thrombospondin, collagen I and III, transforming growth factor-beta (TGF-beta), and plasminogen activator inhibitor-1 (PAI-1) mRNA. Treatment with the ANG II receptor antagonist, eprosartan (60 mg. kg(-1).day(-1)), lowered blood pressure (95 +/- 5 mmHg) and proteinuria (19 +/- 8 mg/d) and abrogated the increased TGF-beta, fibronectin, thrombospondin, collagens I and III, and PAI-1 mRNA expression. An increase in left ventricular weight was observed in five-sixths nephrectomized rats (0.13 +/- 0.01 vs. 0.08 +/- 0.01 g/100 g body wt), a response that was inhibited by eprosartan treatment (0.10 +/- 0.01 g/100 g). Left ventricular expression of TGF-beta and fibronectin was also increased in rats with renal disease; however, the small decreases in expression observed in eprosartan-treated rats did not reach statistical significance. These data suggest that eprosartan may be beneficial in progressive renal disease and that the mechanism of action includes inhibition of cytokine production in addition to antihypertensive activity.

Acrylates↗

PubMed2000

Desensitization and resensitization of adrenomedullin-sensitive receptor in rat mesangial cells.

Adrenomedullin is a potent adenylate cyclase activator and a vasodilatory peptide, that has anti-proliferative and apoptotic effects in rat mesangial cells. The present study was designed to determine the mechanisms of desensitization and resensitization of adrenomedullin-sensitive receptor in mesangial cells. Adrenomedullin caused a rapid desensitization of cAMP response evident within 5 min that was almost complete by 1 h of treatment. Pretreatment of cells with forskolin, that activates protein kinase-A by direct activation of adenylate cyclase, also caused adrenomedullin receptor desensitization. In addition, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿], a potent protein kinase-A inhibitor inhibited adrenomedullin-induced desensitization of cAMP response. Adrenomedullin also caused desensitization of isoproterenol- and epinephrine-mediated cAMP accumulation. Furthermore, adrenomedullin induced cross-desensitization of endothelin-stimulated inositol phosphate accumulation. The attenuated cAMP response of adrenomedullin was restored to original levels within 2 h of agonist removal. This resensitization response was blocked by treatment with okadaic acid, a protein phosphatase (protein phosphatase-1/protein phosphatase-2A) inhibitor, during the 2 h resensitization period, indicating that protein phosphatase-1/protein phosphatase-2A may be involved in the resensitization of the adrenomedullin-sensitive receptor. We demonstrate for the first time in rat mesangial cells that the adrenomedullin-sensitive receptor undergoes heterologous desensitization and resensitization, and that it likely involves protein kinase-A and protein phosphatase-1/protein phosphatase-2A, respectively.

Adrenomedullin↗

PubMed2000

Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione¿, a PI3-kinase inhibitor, attenuated only calcitonin gene-related peptide-induced ERK and not P38 MAPK activation. Thus, these data suggest that activation of ERK by calcitonin gene-related peptide involves a H89-sensitive protein kinase A and a wortmannin-sensitive PI3-kinase while activation of p38 MAPK by calcitonin gene-related peptide involves only the H89 sensitive pathway and is independent of PI3 kinase. This also suggests that although both ERK and P38 can be activated by protein kinase A, the distal signaling components to protein kinase A in the activation of these two kinases (ERK and P38) are different.

Androstadienes↗

PubMed2000

Adrenomedullin decreases extracellular signal-regulated kinase activity through an increase in protein phosphatase-2A activity in mesangial cells.

Adrenomedullin is a recently identified peptide hormone that has receptors in a number of different systems including renal mesangial cells. We reported recently that adrenomedullin can cause a decrease in extracellular signal-regulated kinase (ERK) activity and increase jun amino-terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) acitivities in rat mesangial cells. Associated with these responses we also reported that adrenomedullin can decrease proliferation and increase apoptosis in mesangial cells. The major aim of the present study was to examine the mechanism of decrease in ERK activity by adrenomedullin and to identify the role of protein phosphatase 2A (PP2A) in the decrease in ERK activity, using okadaic acid [9,10-Deepithio-9,10-didehydroacanthifolicin], a selective inhibitor of PP2A at low nanomolar concentrations. The adrenomedullin-induced decrease in [3H]-thymidine incorporation and increase in apoptosis were reversed by okadaic acid at the concentration that selectively inhibits PP2A. Okadaic acid completely reversed the ERK inhibition caused by adrenomedullin, suggesting that PP2A may be involved in the adrenomedullin-mediated changes in proliferation, apoptosis and ERK activity. PP2A activity in mesangial cells was increased over time following exposure to adrenomedullin. The tyrosine phosphorylation of ERK did not change significantly following adrenomedullin treatment although the ERK activity was decreased significantly. This suggests that the decrease in ERK activity is not mediated through a decrease in MEK (a dual phosphorylating kinase upstream of ERK) or by an increase in MKP-1/2 (a dual specificity phosphatase) activities. Thus we conclude that the mechanism of adrenomedullin-induced decrease in ERK activity in rat mesangial cells is at least in part mediated by an increase in PP2A activity.

Adrenomedullin↗

PubMed2000

Unmasking of endothelin-1-induced natriuresis and renal vasodilation in the dog by enrasentan (SB 217242).

Infusion of endothelin-1 (ET-1) into conscious, chronically instrumented dogs (1, 3 and 10 ng/kg/min i.v.) resulted in decreases in renal plasma flow, urine flow and sodium excretion. Intravenous ET-1 infusion in the presence of enrasentan at 30 microg/kg/min, resulted in an increase in renal plasma flow and sodium excretion; however, at a higher infusion rate (100 microg/kg/min) no renal vasodilation or natriuresis was observed. These data indicate that enrasentan can unmask ET(B)-receptor-induced renal vasodilation and natriuresis.

Animals↗

PubMed2000

Apoptosis in polycystic kidney disease: involvement of caspases.

Polycystic kidney disease (PKD) is characterized by the development of large renal cysts and progressive loss of renal function. Although the cause of the development of renal cysts is unknown, recent evidence suggests that excessive apoptosis occurs in PKD. With the use of terminal deoxynucleotidyl transferase dUTP nick-end labeling staining, we have confirmed the presence of apoptotic bodies in cystic kidneys of congenital polycystic kidney (cpk) disease mice carrying a homozygous mutation at 3 wk of age. Apoptosis was localized primarily to the interstitium with little evidence of cell death in cyst epithelium or noncystic tubules. In addition, we observed that the expression of various caspases, bax and bcl-2, was upregulated in cystic kidneys. With the use of various substrates in enzyme activity assays, we have demonstrated a greater than sevenfold increase in caspase 4 activity and a sixfold increase in caspase 3 activity. These data suggest that there is a caspase-dependent apoptosis pathway associated with PKD and support the hypothesis that apoptotic cell death contributes to cyst formation in PKD.

Animals↗

PubMed2000

Renal vasopressin receptor expression and function in rats following spaceflight.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.

Animals↗

PubMed1999

Cloning and functional characterization of a sodium-dependent phosphate transporter expressed in human lung and small intestine.

A cDNA clone with 53% amino acid identity to the human type II sodium-dependent phosphate transporter (NaPi-3) was isolated from human small intestine and lung. Functional characterization in Xenopus laevis oocytes showed this cDNA to encode a sodium-dependent phosphate transporter. The electrogenic response is similar to that found in other type II transporters but an inverse pH dependence was observed. By Northern blot, a 4.2-kb transcript was found to be abundantly expressed in lung and, to a lesser degree, in several other tissues of epithelial origin including small intestine, pancreas, prostate, and kidney. This transcript encompasses a 2.073-kb open reading frame which is most closely related (78% amino acid identity) to the mouse sodium-dependent phosphate transporter IIb isoform. This novel transporter, designated human NaPi-3b (Genbank AF111856), appears to be an isoform of the mammalian renal type II co-transporter family.

Amino Acid Sequence↗

PubMed1999

Regulation of glomerular mesangial cell proliferation in culture by adrenomedullin.

Adrenomedullin is a recently discovered vasodilatory peptide that has been shown to be a potent activator of adenylate cyclase in a variety of cell systems, including rat mesangial cells. The major aim of the present study was to determine the regulation of rat mesangial cell proliferation (using [3H]thymidine incorporation as an index), apoptosis (using nucleosome-associated cytoplasmic DNA fragmentation as an index) and mitogen-activated protein kinase (MAPK) cascade, specifically extracellular signal-regulated kinase (ERK), jun-amino terminal kinase (JNK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, by adrenomedullin-stimulated cyclic AMP-protein kinase-A pathway. Adrenomedullin increased cAMP levels significantly above basal and the response was inhibited by the adrenomedullin receptor antagonist, adrenomedullin-(22-52). Adrenomedullin also decreased [3H]thymidine incorporation and increased nucleosome-associated cytoplasmic DNA fragmentation, in a concentration-dependent fashion. Both these responses were receptor mediated as, adrenomedullin-(22-52) inhibited these effects. The decrease in proliferation and increase in apoptosis were both mimicked by forskolin, a direct adenylate cyclase activator. Adrenomedullin-mediated decrease in proliferation and increase in apoptosis were inhibited by H89 [[N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride]], a potent protein kinase-A inhibitor. Associated with the changes in proliferation and apoptosis, adrenomedullin decreased ERK2 activity, and increased JNK1 and P38 MAPK activities. All these kinase activities, except the increase in JNK1 activity could be simulated using forskolin. In addition, only adrenomedullin-mediated changes in ERK2 and P38 MAPK activities were inhibited by H89 while, adrenomedullin-stimulated JNK1 was not consistently inhibited by the protein kinase-A inhibitor. These results suggest that adrenomedullin might play an important role in mesangial cell turnover and that although adrenomedullin-mediated responses are primarily cAMP-dependent, it does not preclude the involvement of cAMP-independent pathways.

Adrenomedullin↗

PubMed1999

SB203580 reverses adrenomedullin's effect on proliferation and apoptosis in cultured mesangial cells.

Adrenomedullin is a potent vasodilatory peptide that has a variety of effects in a number of different systems including kidney. In cultured rat glomerular mesangial cells adrenomedullin increases cAMP, decreases proliferation and increases apoptosis. Associated with the anti-proliferative and apoptotic effects, adrenomedullin also causes a decrease in extracellular signal-regulated kinase2 (ERK2) and an increase in cJun N-terminal kinase 1 (JNK1) and P38 mitogen-activated protein kinase (P38 MAPK) activities. The purpose of the present study was to examine the role of P38 MAPK on adrenomedullin-mediated inhibition of [3H]thymidine incorporation (an index of proliferation) and on adrenomedullin-stimulated nucleosome-associated cytoplasmic DNA fragmentation (an index of apoptosis) in mesangial cells, using a selective inhibitor of P38 MAPK, SB203580 [[4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-im idazole], and also to characterize the proximal signal transduction pathways of the three MAPKs in relation to [3H]thymidine incorporation and cytoplasmic DNA fragmentation using a phosphotidyl inositol-3-kinase inhibitor, wortmannin [[1S-(1alpha,6b alpha,9alphabeta,11alpha,11b beta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11,11b-octahydro-1-(methoxyme thyl)-9a,11b-dimethyl-3H-furo[4,3,2-de]indeno[4,5-h]-2-benzopyran-3,6,9- trione]. SB203580 significantly reversed the effects of adrenomedullin on [3H]thymidine incorporation and cytoplasmic DNA fragmentation, and inhibited only P38 MAPK activity. It had no effect on ERK2 and JNK1 activities. Wortmannin, on the other hand, inhibited only adrenomedullin-stimulated cytoplasmic DNA fragmentation and did not affect adrenomedullin-mediated inhibition of [3H]thymidine incorporation. Wortmannin also inhibited adrenomedullin-stimulated P38 MAPK activity without affecting ERK2 and JNK1 activities. These results indicate that: (a) In rat mesangial cells adrenomedullin-mediated inhibition of [3H]thymidine incorporation and stimulation of nucleosome-associated cytoplasmic DNA fragmentation are sensitive to SB203580, and (b) adrenomedullin activates a P38 MAPK through a wortmannin-sensitive kinase. The data using SB203580 suggest an important physiological role for P38 MAPK in rat mesangial cell proliferation and apoptosis.

Adrenomedullin↗