Evaluation of the metabolic changes during hemodialysis by signal averaged ECG.

Cardiovascular diseases are frequent complications of end-stage kidney disease. The aim of the present study was to prove the arrhythmogenic effect of dialysis using signal averaged ECG. The ECG changes and laboratory parameters (sodium, potassium, urea and creatinine levels) were detected during hemodialysis treatment in 26 patients suffering from end-stage kidney disease. The tests and the ECG were performed four times, before (0. minute), during (at 15 and 90 min), and eventually after dialysis (at 240 min). The duration of the QRS complex, high-frequency low-amplitude signals (HFLA), and root-mean-square voltage of the terminal 40 ms of the filtered QRS (RMS) were determined. We considered test results to be positive when two of the three tested parameters were outside the normal range: QRS > 120 ms, RMS < 20 uV, HFLA > 39 ms. Signal averaged ECG was positive in two cases (8%) before and after the dialysis. The duration of the QRS-complex increased significantly during the dialysis (predialysis: 109 +/- 7.6 ms, postdialysis: 116 +/- 8.0 ms, p < 0.0001). Serum urea nitrogen (predialysis: 26.2 +/- 5.4, postdialysis: 11.4 +/- 3.3 mmol/l, p <0.0001) and serum creatinine levels (predialysis: 931 +/- 212, postdialysis: 434 +/- 120 micromol/I, p < 0.0001) decreased significantly during the treatment. Significant and continuous decrease in the potassium levels were detected (predialysis: 5.30 +/- 0.72, postdialysis: 3.91 +/- 0.42 mmol/I, p < 0.0001) during the dialysis. Serum sodium levels (predialysis: 139 +/- 2.7, postdialysis: 141.4 +/- 2.2 mmol/I) had not changed during the dialysis. A significant negative correlation was found between decreasing potassium levels and increasing QRS duration (r = - 0.48, p = 0.01). Our results support our primer assumption that the metabolic changes during dialysis treatment can lead to considerable risk of cardiac arrhythmias.

Plaque volume derived from three-dimensional reconstruction of coronary angiography predicts the fractional flow reserve.

OBJECTIVES: To compare the data calculated from the three dimensional (3D) reconstruction of a coronary stenosis with the fractional flow reserve (FFR) values measured on the same coronary segment. METHODS: Multiple projections of 22 patients (7 female, 15 male, age: 61 ± 9.73 years) were evaluated by the IC30 software of the Axiom Artis X-ray machine. 3D reconstruction was successfully carried out on 23 coronary arteries (14 LAD, 4 CX and 5 RCA). RESULTS: Regression analysis demonstrated significant relationship between the cross-sectional area percentage stenosis (AS) calculated based on the 3D measurement and the FFR (r: -0.566, p: 0.008), as well as between the 3D derived plaque volume (PV) and the FFR (r: -0.501, p: 0.018). On the other hand, the diameter stenosis (DS) and the minimal lumen diameter (MLD) did not correlate with the FFR values. According to the Receiver Operating Characteristic (ROC) analysis the rank of the areas under the ROC curves (AUC) was the following: 1. PV (0.76), 2. AS (0.74), 3. DS (0.62), 4. MLA (0.55), and 5. MLD (0.51). The difference between the AUC of the PV and MLA was found to be significant (p=0.02). The best agreement with the FFR was found when the PV was >44% (sensitivity 66.67%, specificity 82.35%) and the 3D AS was >60% (sensitivity 100%, specificity 47%). CONCLUSION: Besides the 3D AS the calculated PV characterizing the entire lesion is also an important predictor of the flow consequence of the stenosis.

Executive function in deficit schizophrenia: what do the dimensions of the Wisconsin Card Sorting Test tell us?

Neuropsychological characterization of the schizophrenia deficit syndrome is an unresolved issue. The initial assumption was that patients with deficit syndrome show more definitive impairments on tests sensitive for frontal and parietal functions compared with nondeficit patients,but recent studies failed to confirm this assumption. The fundamental question is whether a more refined delineation of executive dysfunctions is able to yield differences between deficit and nondeficit patients. To investigate this question, we implemented a factor analytic approach to explore potential differences between deficit and nondeficit patients using the Wisconsin Card Sorting Test (WCST). Our paper presents an exploratory factor analysis of the WCST on schizophrenia patients and healthy samples, and a comparison among deficit, non-deficit patients with schizophrenia and control samples using the identified factors. A total of 154 patients with schizophrenia fulfilling the criteria for the deficit syndrome, 121 nondeficit patients, and 130 healthy controls were compared. Factor analysis of the WCST variables using the principal component method resulted in a two-factor solution. Comparison of the diagnostic groups on each of the factors revealed that deficit schizophrenia patients suffer from a more severe degree of impairment on the 'General executive function' factor than nondeficit schizophrenia patients. To our knowledge this is the first study that compared patients with the deficit and non-deficit forms of schizophrenia using WCST factor analytic techniques. Our results provide an insight into the cognitive profile of schizophrenia patients with regard to WCST, which could serve as a framework for future clinical and research endeavors.

Microarray and phosphokinase screenings leading to studies on ERK and JNK regulation of connective tissue growth factor expression by angiotensin II 1a and bradykinin B2 receptors in Rat1 fibroblasts.

Rat1 fibroblasts stably transfected with the rat angiotensin II (AngII) AT1a and bradykinin (BK) B2 receptor cDNAs gained the ability to bind Ang II and BK. Wild-type Rat1 cells bound neither ligand. Exposure to either effector led to characteristic Galphai and Galphaq signal cascades, the release of arachidonic acid (ARA), and the intracellular accumulation of inositol phosphates (IP). Microarray analyses in response to BK or AngII showed that both receptors markedly induce the CCN family genes, CTGF (CCN2) and Cyr61 (CCN1), as well as the vasculature-related genes, Cnn1 and Egr1. Real time PCR confirmed the increased expression of connective tissue growth factor (CTGF) mRNA. Combined sequence-based analysis of gene promoter regions with statistical prevalence analyses identified CREB, SRF, and ATF-1, downstream targets of ERK, and JNK, as prominent products of genes that are regulated by ligand binding to the BK or AngII receptors. The binding of AngII or BK markedly stimulated the phosphorylation and thus the activation of ERK2, JNK, and p38MAPK. A BKB2R and an AT1aR chimera which displayed only negligible G-protein-related signaling were constructed. Both mutant receptors continued to activate these kinases and stimulate CTGF expression. Inhibitors of ERK1/2 and JNK but not p38MAPK inhibited the BK- and AngII-stimulated expression of CTGF in cells expressing either the WT or mutant receptors, illustrating that ERK and JNK participate in the control of CTGF expression in a manner that appears to be independent of G-protein. Conversely, addition of BK or AngII to the cell line expressing WT AT1aR and BKB2R downregulated the expression of collagen alpha1(I) (COL1A1) mRNA. However, these effectors did not have this effect in cells expressing the mutant receptors. Thus, a robust G-protein related response is necessary for BK or AngII to affect COL1A1 expression.

Fatty acid composition of intramuscular lipids in various muscles of Holstein-Friesian bulls slaughtered at different ages.

Holstein-Friesian bulls were slaughtered at 7, 14 and 19 months of age. Samples were collected from the psoas major, longissimus and semitendinosus muscles. The total lipids (TL) of the samples were extracted and the fatty acid compositions were analysed by gas chromatography. Both the slaughtering age and the type of muscles had significant effects on the intramuscular TL contents and fatty acid compositions. The longissimus muscle had higher intramuscular TL both at 14 and 19 months than at 7 months of age. As the bulls became older the proportion of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) increased in the TL of each muscle tested, whereas that of polyunsaturated fatty acids (PUFA) decreased. Among the muscle types, the semitendinosus had the lowest, intramuscular TL at each slaughtering age and the psoas major the highest. Except for SFA at 7 months of age, the semitendinosus showed lower levels of SFA and MUFA and higher proportions of PUFA than the other two muscles.

The epidemiology of hypertension and its associated risk factors in the city of Debrecen, Hungary.

The purpose of this work was to estimate the prevalence of hypertension and cardiovascular risk factors and its association with sociodemographic, behavioural and lifestyle characteristics among the adult population of the city of Debrecen, Hungary.A cross-sectional study was conducted in 1996. Amongst 21 800 inhabitants aged 30-65 y risk screening for cardiovascular disease (CVD) was estimated by a questionnaire that included sociodemographic, lifestyle characteristics, family history of CVD and results of self-reported data of body weight, height and blood pressure. Of the total of 19 961 surveyed sample, 37.02% were considered to be hypertensive, 53.73% were overweight, 32.18% were current smokers and 58.85% were physically inactive. Hypertensives were older than normotensives (50.81+/-9.01 vs 44.78+/-8.97 y, P<0.001). The prevalence of various risk factors amongst hypertensives as compared to normotensives were overweight (68.49 vs 45.06%, P<0.0001), current smoking (28.38 vs 34.41%, P<0.0001), physical inactivity (64.78 vs 55.36%, P<0.001), and high alcohol consumption (1.91 vs 1.06%, P<0.01). Of the hypertensives 37.11% were on drug therapy. Of those on therapy, only 17.03% had normal blood pressure. Being overweight and having low physical activity was positively associated with hypertension (OR=2.25, CI=2.11-2.40) and (OR=1.26, CI=1.15-1.38). Manual work, a family history of CVD, low education, and the male gender were also associated with hypertension and more CVD risk factors. These findings illustrate a very high prevalence of hypertension and CVD risk factors in Debrecen, indicating the importance of the need for more effective prevention programmes and control of hypertension in Hungary.

[Epidemiology of hypertension in adolescence and factors influencing blood pressure]. / A serdülókori hypertonia epidemiológiája és a vérnyomást befolyásoló tényezók.

The authors summarize the determining and influencing factors of adolescent hypertension. An overview of the definition and prevalence of hypertension in adolescence is given and the predictive role of the adolescent hypertension on the incidence of adult cardiovascular diseases is pointed out. According to the previous literature data, adult hypertension is more frequent in those people who have had hypertension in their adolescence. There are no widely used, population-based nomograms of adolescent hypertensives available. According to the opinion of the authors, a population-based hypertension screening program would help in delineating the factors influencing adolescent blood pressure, and the most frequent risk factors for hypertension in Hungary. With the follow-up and appropriate treatment of the hypertensives the reduction of target-organ damages may be possible.

Role of hydroxyl containing residues in the intracellular region of rat bradykinin B(2) receptor in signal transduction, receptor internalization, and resensitization.

In past reports we illustrated the importance of Y131, Y322, and T137 within the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) for signal transduction and receptor maintenance (Prado et al. [1997] J. Biol. Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555). In this report, we mutate the remaining hydroxyl possessing residues located within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) did not affect BK activated phosphatidylinositol (PI) turnover or receptor internalization. Chimeric exchange of the last 34 amino acids of BKB2R C-terminus with the corresponding 34 amino acids of the rat angiotensin II AT1a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant with normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR fragment (326-342), resulted in a receptor with a retarded internalization but a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V showed little change in receptor uptake but a pronounced loss of BK activated PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in very poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S348A led to poor internalization; whereas the retention of S348 and mutation of S341A resulted in a receptor with a much greater internalization than WT. These and other results suggest that the presence of S348 promotes internalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate that the distal C-terminus including its S/T cluster is important for both rBKB2R internalization and signal transduction. Individual S/T residues within this cluster appear involved in either signal transmission or receptor uptake capacity. However, replacement of the entire distal tail region with the corresponding rAT1aR sequence, also containing an S/T cluster, enables the BKB2R/AT1aR chimera to act in a very similar manner to wild type rBKB2R.

A tryptamine analog with high affinity to the heart tissues is a potential antiarrhythmic agent.

A novel tryptamine analog, 1-methyl-3-[N-(3-indolyl)ethyl]carbamoyl-1,4-dihydropyridine (T-CDS) was synthesized and converted into a stable, solid complex with 2-hydroxypropyl-beta-cyclodextrin. An aqueous solution of the complex was given intravenously to dogs and the concentration of T-CDS and its corresponding quaternary (T-Q+) forms were monitored in the blood for 50 min. The effect of the drug on vital heart parameters was monitored throughout the studies. At the end of the experiment the dogs were sacrificed and the concentration of the quaternary pyridinium form (T-Q+) was determined in the different heart tissues, as well as in the kidney, liver, lung, brain, urine and cerebrospinal fluid. The compound was found to be selectively bound to the heart muscles and showed different concentrations in different heart tissues. The T-Q+ concentrations were much higher in the heart after administration of the dihydro form (T-CDS), than after administering T-Q+ directly. The compound was found to be active on certain vital signs of the cardiovascular system and could be an effective and safe antiarrhythmic agent.

p53 down-regulates human bradykinin B1 receptor gene expression.

The tumor suppressor, p53, has been shown to transcriptionally activate or silence a number of target genes. As an activator, p53 relies on its specific consensus sequence within the promoter. It is not clear whether p53 requires a specific DNA binding site in its action as a gene repressor. This report demonstrates that the human BKB1R gene is a p53 target. Expression of p53 in transiently transfected SV40-transformed IMR90 cells strongly suppressed luciferase reporter activity driven by a 1.8 kb BKB1R promoter as well as its minigene. These down-regulations were p53 dose-dependent. p53 reduced both basal and induced promoter activities of the minigene. Expression of p53 abolished the inducibility of the minigene. Induction of endogenous p53 expression by etoposide also inhibited promoter activity and minigene inducibility. Replacing the region containing both the putative p53 binding site and the TATA-box with a basal adenovirus promoter in the 1.8 kb promoter construct did not prevent p53 from inhibiting BKB1R promoter activity. Thus suppression by p53 is not mediated by competition with the TATA-binding protein and is not through interaction with the putative p53-binding site. p53 also does not appear to suppress BKB1R gene expression through interaction with c-Jun which functions in the inducibility of this gene [Yang et al., 2001].

Mediator caused induction of a human bradykinin B1 receptor minigene: participation of c-Jun in the process.

The bradykinin B1 receptor (BKB1R) gene is expressed in selected tissues such as lung and kidney. In these tissues it is expressed at a very low level until induced by inflammatory mediators. Our aim has been to understand the mechanism of this regulatory process. A human BKB1R minigene was constructed. It contained a 1.8 kb promoter, the entire exon I, 1.5 kb of intron I, the entire exon II and intron II, and the luciferase gene as a reporter. Transient transfection of the minigene into SV40-transformed IMR90 cells (IMRSV) resulted in a promoter activity which was activated by the mediators, lipopolysaccharide and (LPS) desArg(10)-kallidin. In contrast, these mediators did not induce the activity of the 1.8 kb promoter construct alone. Thus, motifs exclusive of the promoter such as 5'-UTR and/or intron regions are required for mediator-induced expression of this gene. Promoter activities of both the minigene and the 1.8 kb promoter construct were enhanced in a dose-dependent manner upon cotransfection with c-Jun. Furthermore, cotransfecting c-Jun with the minigene achieved the maximal promoter activity with no further increase in response to mediators. Conversely, the induction of the minigene promoter activity by mediators was abolished upon cotransfection with a dominant negative mutant of c-Jun. Other experiments suggest that multiple AP-1 sites are interactive with the c-Jun upregulation of this gene. Taken together, these results point to c-Jun as a key intermediary in the activation of the expression of this gene by mediators. However, participation of motifs outside of the promoter are necessary to obtain this inducible expression.

Prevalence of cardiovascular risk factors of the smokers and non-smokers in the city of Debrecen, Hungary.

The aim of our study was to compare the major cardiovascular disease (CVD) risk factors of smokers and non-smokers. Risk screening of CVD was estimated by a questionnaire, via interview. Random samples of 20 000 inhabitants of Debrecen, Hungary, aged 30-65 y, took part in the study. 19 922 questionnaires were considered appropriate for further evaluation. 32.2% of the participants (n=6410) were smokers, whose data were compared to those of the 68.8% of non-smokers (n=13 512). There were more male smokers than female (39.3% vs 27.7%), (P<0.001). 36.5% of males and 58.9% of females had not previously smoked regularly (P<0.001). 24.2% of males and only 13.3% of females were able to stop smoking (P<0.001). 8.7% of the participants smoked more than 20 cigarettes per day (14.8% of males, 5.0% of females), (P<0.001). Smokers were younger, with a mean age of 43.4 y vs 47.1 y for non-smokers (P<0.01). The ex-smokers and non-smokers had a higher body mass index than light, moderate and heavy smokers (26. 75+/-4.1 kg/m2 and 26.09+/-4.3 kg/m2 vs 24.87+/-3.9 kg/m2 and 24. 89+/-4.2 kg/m2 and 25.32+/-4.3 kg/m2, respectively), (P<0.001). The results of the last measured blood pressures did not differ between the two groups. 94.8% of smokers and 93.6% of non-smokers did not perform any regular leisure time exercises (P<0.01). 39.8% of smokers regularly ate fatty food, in comparison to 28.0% of non-smokers (P<0.001). 30.6% of smokers vs 28.6% of non-smokers were factory workers while 69.4% of smokers vs 71.4% of non-smokers did sedentary jobs (P<0.001). 2.3% of smokers vs 0.9% of non-smokers admitted regular consumption of alcohol (P<0.001). Amongst the parents and brothers/sisters of smokers the prevalence of heart attack was higher 19.7% vs 18.7%, than for those of non-smokers (P<0. 05). We observed an accumulation of cardiovascular risk factors in the case of smokers, which indicates the higher susceptibility of smokers to CVD.

Regulation of inducible bradykinin B1 receptor gene expression through absence of internalization and resensitization.

Rapid induction and down-regulation of bradykinin B1 receptor (BKB1R) gene expression is tightly regulated at the transcriptional and mRNA levels (Zhou et al. [1998] Biochem. J. 330:361-366; Zhou et al. [1999] Mol. Cell Biol. Res. Commun. 1:29-35). Here we explore regulation of BKB1R expression at the protein level. To make this inducible gene express constitutively, we utilized a bicistronic mammalian expression vector (pCMin) for stable transfection of the BKB1R gene into human lung fibroblasts, IMR90SV40. The BKB1R displayed a high affinity and specificity (K(d) = 0.5 nM) for desArg(10)-kallidin. The receptor mediated such signaling events as arachidonic acid (ARA) release, phosphoinositide (PI) turnover and Ca(2+)-flux. The receptor function proved differentially desensitized. For example, after initial exposure to desArg(10)-kallidin, a second stimulation with desArg(10)-kallidin did not induce further Ca(2+)-flux or ARA-release while PI-turnover continued unabated. Unlike most of the G-protein coupled receptors, the BKB1R did not internalize within 60 min of exposure to 10 nM desArg(10)-kallidin. It also did not resensitize. Thus, the duration and signal capacity of the BKB1R at the protein level is regulated through lack of internalization, an absence of resensitization and a lack of desensitization for certain events such as PI turnover. In fact, the absence of BKB1R resensitization is likely a very important contributor to the rapid disappearance of this inducible receptor.

Effect of the G-protein, G alpha(i2), and G alpha(i3) subunit knockdown on bradykinin-induced signal transduction in rat-1 cells.

The bradykinin (BK) B2 receptor (BKB2R) has been shown to interact with the G alpha(q) subunit family. However, it has remained unclear whether this receptor also interacts with the G alpha(i) subunit family. To further resolve this issue, two antisense expression plasmids were generated. In these, the 5'-untranslated regions of rat G alpha(i2) and G alpha(i3) cDNAs were used as specific antisense templates. The plasmids were transfected into Rat-1 cells, which expressed a stably transfected rat BKB2R cDNA and bound BK with a Kd of approximately 3 nM. In these cells, the transfected BKB2R was fully linked to inositol phosphate production, arachidonic acid (ARA) release, and Ca2+ flux. A number of cell lines, each a G alpha(i2) or G alpha(i3) knockdown, were isolated. Of these, two cell lines were chosen for study. One, designated 2-E3, displayed over a 70% decrease in the expression of G alpha(i2) without a change in the expression of G alpha(i3) or G alpha(q). Another, 3-G9, exhibited over a 70% decrease of G alpha(i3) protein without a change in G alpha(i2) or G alpha(q) expression. Knockdown of either G alpha(i2) or G alpha(i3) protein production did not affect the binding of bradykinin. In the G alpha(i2)-depleted 2-E3 cells, BK induced ARA release was reduced by more than 60%. Interestingly, the production of total inositol phosphate in response to BK was also reduced by approximately 35%. However, G alpha(i2) knockdown had no significant effect on BK-induced Ca2+ influx. In the G alpha(i3)-depleted 3-G9 cells, BK-induced ARA release was decreased by over 50%. In this case [Ca2+]i increase in response to BK was reduced by over 50%. This knockdown also resulted in reduced BK-activated total inositol phosphate production. Moreover, cAMP augmented the BK-induced ARA release. Depletions of G alpha(i2) and G alpha(i3) further enhanced this cAMP-dependent BK induction of ARA release. Taken together, these results delineate direct interaction of the BKB2R with both G alpha(i2) and G alpha(i3) subunits in addition to the heretofore described interaction of BKB2R with the G alpha(q) subunit family.

Posttranscriptional destabilization of the bradykinin B1 receptor messenger RNA: cloning and functional characterization of the 3'-untranslated region.

We showed previously that the inducible bradykinin B1 receptor (BKB1R) gene expression is regulated, in part, through mRNA stabilization. Here we clone the 3'-untranslated region (3'-UTR) of the BKB1R. This region proves to be very short, containing only 14 bases with an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon. Reverse transcription confirms the presence of this alternative polyadenylation signal. Northern blot shows a single species of BKB1R mRNA of approximately 1.4 kb in agreement with its calculated length. The BKB1R mRNA induced by TNFalpha, phorbol ester, bradykinin, and desArg10-kallidin contain the same 3'-UTR species. To test the role of this region in the regulation of mRNA stability, we generated a chimeric luciferase construct containing the BKB1R 3'-UTR. The mRNA transcribed from the wild-type luciferase gene displayed a half-life of approximately 6 h. The mRNA transcribed from the chimeric construct displayed a half-life of only 1 h. This decrease was also reflected at the level of enzyme activity. Luciferase activity from cells transfected with the chimeric construct was 10 times less than from cells transfected with wild-type luciferase. The data presented provide compelling evidence that the 3'-UTR is participating in the regulation of BKB1R mRNA stability and its ultimate expression.

[Prevention of atrial fibrillation by atrial-based pacemaker implantation]. / A pitvari fibrilláció megelözése pitvari vagy pitvar-kamrai szekvenciális pacemaker-beültetéssel.

A large number of studies have demonstrated the long term disadvantage of single lead ventricular pacing in sick sinus syndrome. Ventricular pacing mode predicts chronic atrial fibrillation in patients with preimplant paroxysmal atrial fibrillation. The goal of the report was to study the effectiveness of single atrial and dual chamber (atrio-ventricular sequential) pacemaker treatment in the prevention of atrial fibrillation for patients with sick sinus syndrome complicated with paroxysmal atrial fibrillation. In our university hospital 16 atrial based 5 and dual chamber 11 pacemaker were implanted for treatment of patients with sick sinus syndrome (with or without AV conduction disturbances) complicated with paroxysmal atrial fibrillation. The mean age were 61 (24-78), nine males and seven females. Before or during pacemaker implantation sinus node and AV node function analysis, and echocardiography were performed. There were no surgical complications, lead and/or generator failure. All patients had routine follow-up performed at 4 weeks, 3 months, 6 months. Mean follow up was 31 +/- 8 months (range 3 to 93 months). The atrial based and dual chamber pacing were effective in 90% of our cases. In one patient the treatment had to be combined with propafenone. According to our result, the atrial based pacing may be used to reduce the incidence of atrial fibrillation with careful programming of the base atrial pacing rate, and it is associated with lower frequencies of thromboembolic complications and pacemaker syndrome.

Motif mutation of bradykinin B2 receptor second intracellular loop and proximal C terminus is critical for signal transduction, internalization, and resensitization.

In the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr131 and Tyr322) to signal transduction and receptor internalization (Prado, G. N., Taylor, L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638-14642). Here, we investigate the effect of exchanging both tyrosine residues simultaneously for alanine, phenylalanine, or serine, termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S) receptors, respectively. All of these mutants bound bradykinin (BK) normally, with a Kd of approximately 1.1 nM. However, although phosphoinositide (PI) turnover in response to BK by Y131A and Y131S proved negligible, the YAYA mutant returned BK-activated PI turnover to wild type (WT). In contrast, PI turnover with YSYS remained unresponsive to BK. Importantly, the pattern of BK-activated arachidonate release differed markedly in the mutant receptors. For example, whereas Y131S ablated BK-activated arachidonic acid release, conversion of this mutant to YSYS returned the BK-activated receptor function to a level above that of WT. However, YAYA showed only a partial recovery from the poor BK response of Y131A. These and additional results suggest that Tyr131 and Tyr322 interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr137 is functioning as a switch in termination of signal transduction and the initiation of internalization.

Role of EP2 receptors and cAMP in prostaglandin E2 regulated expression of type I collagen alpha1, lysyl oxidase, and cyclooxygenase-1 genes in human embryo lung fibroblasts.

In a recent communication, we demonstrated that prostaglandin E2 (PGE2) lowers basal while it ablates interleukin-1beta((IL-1beta) and transforming growth factor-beta (TGFbeta) upregulated lysyl oxidase (LO) mRNA levels. Correspondingly, PGE2 increases cyclooxygenase-1 (COX1) mRNA in diploid, human embryo lung fibroblasts (IMR90) [Roy et al., 19961. We now report that these actions by PGE2 are routed through cAMP via the PGE2, EP2 receptor. Among the PGE2 receptor types, the IMR90 predominantly express the EP2 mRNA. These cells also express EP3 and EP4 mRNA at comparatively low levels. Northern blot analyses show that 11-deoxy PGE1, an EP2/EP4 agonist, emulates the action of PGE2. In a similar manner to PGE2, 11-deoxy PGE1 decreases basal and TGF-beta induced type I collagen alpha1 (COL) mRNA, basal and IL-1beta induced LO mRNA while it increases COX1 mRNA. Sulprostone, an EP3/EP1 agonist, has no effect on the expression of these three genes. Forskolin, an adenylate cyclase activator, acts in a very similar manner to PGE2 or 11-deoxy PGE1. It suppresses both basal and TGF-beta induced COL mRNA levels. Both PGE2 and 11-deoxy PGE1 increase cAMP to a level comparable with forskolin. The role of the EP2 receptor in controlling collagen production is further underscored in the immortalized Rat-1 fibroblasts, derived from Fischer rat embryos, which do not express detectable EP2 mRNA. In these cells, PGE2 has little effect on COL mRNA level, whereas forskolin increases it. Furthermore, forskolin increases cAMP level in Rat-1 cells, whereas PGE2 does not. Overall, these results illustrate that much of the PGE2 action on the expression of COL, LO, and COX1 genes is mediated through the EP2 receptor and a subsequent increase in intracellular cAMP.

Mechanisms in the transcriptional regulation of bradykinin B1 receptor gene expression. Identification of a minimum cell-type specific enhancer.

To investigate the mechanisms of bradykinin B1 (BKB1) receptor gene expression, transient DNA transfection analyses of human BKB1 receptor gene promoter were performed in SV-40 transformed IMR90 cells. A positive regulatory element (PRE) located at position -604 to -448 base pair (bp) upstream of the transcription start site consistently exhibited, by far, the highest level of relative luciferase activity. A negative regulatory element, at position -682 to -604 bp, was able to completely ablate the function of the PRE. Transfection combined with deletion and mutation analyses illustrated that the PRE contains a classic, powerful enhancer. This enhancer was minimized to a 100-bp element at position -548 to -448 bp. A 78-bp fragment of negative regulatory element functioned as a silencer. Transient transfection of the enhancer construct, driven by heterologous herpes simplex thymidine kinase promoter, into a variety of cell types, showed that this enhancer presents a cell-type specific feature. In the characterization of the enhancer, motifs A (-548 to -532) and B (-483 to -477) were found to be essential for full enhancer activity. Motif D (-472 to -467) played a smaller role in enhancer activation. Gel shift and antibody supershift assays determined that an AP-1 factor binds with motif B. The nuclear protein which binds to motif A has yet to be identified. Both factors are the critical regulators for this enhancer activation.

Roles for interleukin-1beta, phorbol ester and a post-transcriptional regulator in the control of bradykinin B1 receptor gene expression.

Bradykinin B1 receptor (BKB1R) is involved in a variety of pathophysiological processes, particularly those related to inflammation. The gene for this receptor is known to be upregulated by interleukin (IL)-1beta, a proinflammatory cytokine. However, the molecular mechanisms involved in the regulation of the BKB1R gene expression have not been defined. We demonstrated that IL-1beta induces a rapid increase in BKB1R mRNA level and the binding of desArg10-kallidin in human embryo lung fibroblasts (IMR90). This increase in BKB1R mRNA level is protein synthesis-independent as indicated by treatment of cells with cycloheximide (CHX) or puromycin (PUR). By testing the IL-1beta effect on BKB1R mRNA degradation, we showed that the IL-1beta upregulation of BKB1R expression is achieved through both transcriptional activation and post-transcriptional mRNA stabilization. In addition to the IL-1beta effects, translation inhibitors, CHX and PUR increase the steady state BKB1R mRNA level by inhibiting BKB1R mRNA degradation. Removal of the CHX block with subsequent resumption of protein synthesis results in a sizable increase of desArg10-kallidin binding. Using signalling pathway inhibitors, we show that IL-1beta functions through a protein tyrosine kinase, not protein kinase C or protein kinase A. However, activation of protein kinase C by phorbol 12-myristate 13-acetate increases the level of BKB1R mRNA and the binding of desArg10-kallidin. This increase is blocked by NF-kappaB activation inhibitors.