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Corin Gene Minor Allele Defined by 2 Missense Mutations Is Common in Blacks and Associated With High Blood Pressure and Hypertension
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Background— The natriuretic peptide system contributes to blood pressure regulation. Atrial and brain natriuretic peptides are cleaved into smaller biologically active molecules by corin, a transmembrane serine protease expressed in cardiomyocytes.
Method and Results— This genotype-phenotype genetic association study included replication samples and genomic control to correct for population stratification. Sequencing of the human corin gene identified 2 nonsynonymous, nonconservative single nucleotide polymorphisms (Q568P and T555I) in near-complete linkage disequilibrium, thus describing a single minor I555 (P568) corin gene allele. This allele was present in the heterozygote state in &12% of blacks but was extremely rare in whites (<0.5% were homozygous for the minor allele). In our primary population sample, the Dallas Heart Study, after adjustment for potential confounders, including population stratification, the corin I555 (P568) allele remained independently associated with increased risk for prevalent hypertension (odds ratio, 1.63; 95% CI, 1.11 to 2.38; P=0.013). The corin I555 (P568) allele also was associated with higher systolic blood pressure in subjects not using antihypertensive medication in unadjusted (133.7±20.7 versus 129.4±17.4 mm Hg; P=0.029) and adjusted (132.5±1.6 versus 128.9±0.6 mm Hg; P=0.029) analyses. The independent association of the minor corin allele with increased risk for prevalent hypertension was confirmed in the Multi-Ethnic Study of Atherosclerosis (odds ratio, 1.50; 95% CI, 1.09 to 2.06; P=0.014). In addition, the association of the minor corin I555 (P568) allele with higher systolic blood pressure was confirmed in adjusted analysis in the Chicago Genetics of Hypertension Study (125.8±1.9 versus 121.4±0.7 mm Hg; P=0.03).
Conclusions— The corin I555 (P568) allele is common in blacks and is associated with higher blood pressure and an increased risk for prevalent hypertension.
Received December 12, 2004; de novo received June 14, 2005; revision received July 21, 2005; accepted July 25, 2005.
The endogenous cardiac hormonal system refers to the ability of the heart to synthesize and secrete atrial and brain natriuretic peptide (ANP and BNP) into the circulation in response to various hemodynamic stimuli, primarily myocyte stretch.1 Both ANP and BNP defend against hypertension by vasodilatation2 and natriuresis,3 as well as by opposing the actions of both the renin-angiotensin-aldosterone4 and sympathetic nervous systems.5 In mouse models, overexpression of ANP or its receptor has been shown to cause a "dose-dependent" fall in arterial blood pressure6; selective disruption of the ANP gene (ANP−/−) or the natriuretic peptide receptor (NPRA−/−)7,8 has been shown to cause chronic murine hypertension. Whether loss-of-function mutations in the natriuretic peptide pathways contribute to some cases of human hypertension has not been determined.
Corin is a recently identified transmembrane serine protease that is highly expressed in cardiomyocytes and cleaves inactive pro-ANP and pro-BNP into smaller biologically active molecules.9–11 Here, we describe a corin allele defined by the presence of 2 nonsynonymous, nonconservative (type 1) single nucleotide polymorphisms (SNPs) in near-complete linkage disequilibrium that is enriched in blacks and associated with increased risk for prevalent hypertension and higher blood pressure.
Study Populations
Dallas Heart Study
The primary study sample was the Dallas Heart Study (DHS), a multistep probability-based sample of Dallas County residents 18 to 65 years of age. The rationale, design, and sample validation of the DHS have been published.12 Briefly, race/ethnicity was self-assigned by the participants from the structured list of categories used in the Third National Health and Nutrition Examination Survey.13 Blacks were oversampled to ensure a final representation of 50%. Data were collected in 3 sequential visits: Visit 1 (n=6101) was a household interview conducted by trained field interviewers; visit 2 (n=3398) was a home visit in which blood and urine specimens were obtained; and visit 3 (n=2971) was a visit at the University of Texas Southwestern Medical Center during which imaging tests were obtained. We limited our analysis to persons in the DHS who were ≥35 years of age to increase the positive predictive value of the diagnosis of prevalent hypertension.14 This restricted group included most (76%) of the DHS black participants.
Multi-Ethnic Study of Atherosclerosis
The Multi-Ethnic Study of Atherosclerosis (MESA) was initiated in July 2000 to investigate the prevalence, correlates, and progression of subclinical cardiovascular disease in a population-based sample of 6814 men and women 45 to 84 years of age.15 Race/ethnicity was self-assigned. Details of the data collection and quality control have previously been published.15 Unlike the DHS, participants with known cardiovascular disease (eg, previous history of myocardial infarction or heart failure) were excluded. However, a prior diagnosis of hypertension or treatment for hypertension was not an exclusion criterion.
Chicago Genetics of Hypertension Study
The Chicago Genetics of Hypertension Study is a population-based sample of black subjects from metropolitan Chicago that comprised one of the cohorts in the International Collaborative Study on Hypertension in Blacks.16,17 Race was ascertained by self-report. Unlike the DHS and MESA, a self-reported history of hypertension or treatment for hypertension was an exclusion criterion. Participants from metropolitan Chicago were recruited from the top and bottom quartiles of body mass index. These individuals then collected 3 consecutive 24-hour urine samples that were assayed for sodium and potassium. The final sample consisted of &200 subjects in each of the bivariate extreme quartiles for body mass index and 24-hour urine sodium excretion.
Blood Pressure Measurement
Dallas Heart Study
A total of 5 independent blood pressure measurements were taken in each individual in the seated position at each of the 2 in-home visits by trained and certified personnel using an automated oscillometric device (Welch Allyn, MC) that has been validated against direct arterial pressure measurements.12,18 A total of 5 serial measurements were made with the automated device. The average of the last 3 of the 5 measurements was considered the blood pressure for that visit. For the present analysis, we averaged the blood pressure from the 2 in-home visits. The group means (and blood pressure distributions) were very similar between the interview sample and the phlebotomy and clinic subsamples.12
Multi-Ethnic Study of Atherosclerosis
Formally trained and certified field personnel made blood pressure measurements, and data quality was centrally reviewed by the MESA Data Coordinating Center.15 Resting blood pressure was measured 3 times in subjects in the seated position during a single visit with a Dinamap model Pro 100 automated oscillometric sphygmomanometer (Critikon). The last 2 measurements were averaged.
Chicago Genetics of Hypertension Study
Resting blood pressure was measured at a single home visit in subjects in the seated position with an automated Omron cuff device19 by certified and trained field personnel. A total of 4 measurements were made in each patient in the seated position after resting at least 10 minutes. The recorded blood pressure for each participant was the average of the last 3 measurements made with the automated device. Precision and reproducibility of this automated device have been described.20 The within-subject between-visit reproducibility on a subset was consistently high (R2≥0.82).17
Other Data Collection in the DHS
Coronary Calcium
Two consecutive electron-beam CT scans were performed on each study participant.21 In the present analysis, electron-beam CT scores were classified as coronary artery calcium positive when the mean electron-beam CT score was >10 Agatston units or as coronary artery calcium negative when the mean electron-beam CT score was ≤10 Agatston units as previously reported.21,22
Estimated Glomerular Filtration Rate
Glomerular filtration rate was estimated by the Cockroft-Gault equation23 as follows: glomerular filtration rate=(140−age)/(72×serum creatinine). This value was multiplied by 0.85 to estimate the glomerular filtration rate in women.
Clinical Definitions
In both the DHS and MESA, hypertension was defined according to the Joint National Committee criteria as the presence of 1 of the following: (1) self-reported history of hypertension and current use of any antihypertensive medication and (2) systolic blood pressure ≥140 mm Hg or diastolic blood pressure ≥90 mm Hg.
Diabetes Mellitus
In both the DHS and MESA, diabetes mellitus was defined by 1997 American Diabetes Association criteria as the presence of 1 of the following: (1) a fasting glucose ≥126 mg/dL or (2) a self-reported history of diabetes mellitus and current use of either insulin or an oral diabetes medication.
Sequencing and Genotyping
The corin gene was sequenced in 30 blacks by the UT Southwestern Sequencing Core Laboratory as a component of the Program in Genomic Applications. We focused on exons, the intron/exon boundaries (&30 to 50 nucleotides into the intronic regions), and the 3′- and 5′-flanking regions. The full sequencing results of the corin gene, including all SNPs identified, are available at [email protected] We applied several criteria to prioritize identified SNPs, including a change in amino acid (nonsynonymous), especially those that were biochemically significant (nonconservative) in residues demonstrating conservation among species. Allelic discrimination of the corin variants was accomplished with 5′ nuclease assay (Taqman) and an Applied Biosystems Prism 7900 apparatus. Successful genotype determination was made in ≥99% of subjects in each of the DHS, MESA, and Chicago Genetics of Hypertension Study. Genotyping of markers that are highly informative for ancestry24 was performed with Perlegen Sciences high-density oligonucleotide-hybridization array technology (Perlegen Sciences).25 Genotyping of the DHS black population for these markers demonstrated >96% genotyping success for the 2114 markers.
Statistical Analysis
Statistical analyses were conducted with SAS 9.0 and SAS Genetics 9.0 software. Statistical comparisons of means used the Student t test, assuming unequal variances between the groups when statistically appropriate. Comparisons of proportions between groups used Pearson's χ2 statistic. A 2-tailed probability value was used for all analyses, and a 2-sided probability value of ≤0.05 was considered statistically significant unless otherwise indicated. In multivariate linear and logistic regression analyses in the Dallas Heart Study, we adjusted for the following covariates: Age (continuous variable), gender, body mass index (continuous variable), diabetes, estimated glomerular filtration rate (continuous variable), coronary artery calcium (dichotomous variable), alcohol use, smoking status, and estimated African ancestry (see below). Some of these data were unavailable in our replication cohorts, precluding our ability to adjust for the exact same covariates as in the Dallas Heart Study. In the Multi-Ethnic Study of Atherosclerosis, we were able to adjust for age (continuous variable), body mass index (continuous variable), gender, and diabetes mellitus. In the Chicago Genetics of Hypertension Study, multivariate analysis included adjustment for age (continuous variable), gender, and body mass index (continuous variable). In the Dallas Heart Study and the Chicago Genetics of Hypertension Study, adjusted mean blood pressure parameters were compared between corin genotype groups using adjusted least squares estimates in a generalized linear model. The values for systolic, diastolic, and mean arterial blood pressure are expressed as mean±standard error of the mean.
If subjects are sampled from ≥2 subpopulations or from recently admixed populations such as blacks and Mexican Americans for which the frequencies of marker alleles and disease-susceptibility gene alleles differ, spurious associations may arise.26–28 To allow us to control for such hidden population stratification, we genotyped the black participants in the DHS for 2114 SNPs determined to be highly informative of ancestry24,29,30 (see Sequencing and Genotyping above). This final panel of 2114 SNPs was selected from an initial panel of 2270 SNPs according to the following process: Because European Americans can be considered one of the parental populations for African Americans, we first calculated the pairwise r2 values in every chromosome in the European Americans in the DHS and identified 110 sets of adjacent SNPs in strong linkage disequilibrium (r2>0.9). Strong linkage disequilibrium between adjacent markers can seriously bias the estimation of marker location–specific ancestry; therefore, we retained only 1 SNP from each of these pairs in the subsequent analyses. SNPs that departed significantly from Hardy-Weinberg equilibrium are likely to have been inaccurately genotyped, and those associated with values of P<0.001 were also excluded. The remaining SNPs were examined with a hidden Markov model to estimate the marker allele frequencies in the parental populations. If the estimated allele frequencies at adjacent SNPs in 1 ancestral population are equal to 1.0 and 0.0 but in the other ancestral population are equal to 0.0 and 1.0, it indicates strong linkage disequilibrium between these SNPs, and only 1 of these SNPs was kept for admixture mapping. After these data verification procedures, we defined a final analytic set of 2114 SNPs to use in our statistical methods to adjust for hidden population stratification.
We estimated the marker location–specific ancestry using the hidden Markov model in which the transmission probability was calculated from the continuous gene flow model. This method directly maximizes the likelihood function through an Expectation and Maximization iterative algorithm and allows for the uncertainty of marker allele frequencies in the parental populations (Xiaofeng Zhu, PhD, personal communication, 2005). The ancestry for each individual was estimated with the average ancestry at each marker location across the whole set of SNPs. To adjust for the population stratification, we included this estimated ancestry as a covariate in our linear regression and logistic regression models.
In addition, we applied the genomic control method by Devlin and Roeder31 to adjust for population stratification in association analysis for hypertension status. In this method, an inflation factor (λ) is first estimated using the ancestry marker panel (genomic control markers). We estimated λ as λ=median(Yi2)/0.456, as recommended by Bacanu and colleagues,32 where Yi2, i=1…n, is the χ2 statistic for marker i. Then, an adjusted test statistic Y2/λ is calculated for the tested marker locus. It follows a χ12 distribution under the null hypothesis.
Identification and Prevalence of Corin Gene Sequence Variants
Sequencing of the human corin gene identified 2 nonsynonymous, nonconservative (type 1) SNPs in highly conserved amino acids in exon 12 (Figure 1), corresponding to a cysteine-rich frizzled-like domain. This domain has been identified as critical to the function of the catalytic domain of the protein.33 We genotyped the black participants in the DHS for these 2 SNPs (threonine to isoleucine at amino acid position 555 [T555I], and glutamic acid to proline at amino acid position 568 [Q568P]), which demonstrated that these 2 SNPs were in near-complete linkage disequilibrium. Of the 187 participants in the DHS carrying 1 copy of the P568 allele, 186 also carried 1 copy of the I555 allele. One subject was found to be heterozygous for the Q568P allele but did not carry the I555 allele. The disruption of the linkage disequilibrium in this subject was confirmed by sequencing.
We genotyped a subsample (&20%) of the blacks in MESA for the 2 SNPs (T555I and Q568P). The 2 SNPs demonstrated complete linkage disequilibrium in this sample. The near-complete linkage disequilibrium for the 2 alleles describes in reality a single corin gene minor allele defined by the presence of both SNPs on the same parental chromosome. We refer to this as the minor corin I555 (P568) allele. To reduce genotyping costs and to conserve DNA, we genotyped the remainder of the MESA subjects and the Chicago Genetics of Hypertension sample for the Q568P SNP only. Among blacks in the 3 cohorts (DHS, MESA, and Chicago Genetics of Hypertension), genotyping demonstrated that &10% to 13% of the blacks were heterozygous and 0.5% were homozygous for the corin T555 (P568) minor allele (Table 1). In each of the cohorts, the I555 (P568) allele was in Hardy-Weinberg equilibrium.
Baseline Characteristics Stratified by Corin I555 (P568) Allele
Given the few black homozygotes for the minor corin allele, we grouped them with the heterozygous subjects (corin variant group) and compared them with the remainder of blacks carrying no copy of the minor allele (nonvariant group). The baseline characteristics stratified by corin genotype in the 3 cohorts are shown (Table 2). In the DHS, there were no significant differences between the corin variant and nonvariant groups with regard to demographic and clinical baseline characteristics. However, the mean estimated African ancestry measure (Table 2), derived from the 2114 informative markers (see Methods), was slightly but significantly higher in the corin variant group. Less detailed baseline information was available in MESA and Chicago Genetics of Hypertension, but there were no significant differences between the corin variant and nonvariant groups with regard to age, gender, and body mass index in our 2 validation cohorts or prevalent diabetes in MESA.
Association of Minor Corin Allele With Prevalent Hypertension and Blood Pressure in the DHS
As demonstrated in Figure 2, the prevalence of hypertension was significantly greater in the corin variant compared with the nonvariant group (54.5% versus 46.2%; P=0.03). We applied the genomic control method31 to account for potential confounding from population stratification, and the association of the corin variant group with higher prevalent hypertension remained statistically significant (P=0.03). In multivariate logistic regression analysis that included adjustment for differences in population admixture (Table 3), the corin variant group remained at significantly higher risk for prevalent hypertension (odds ratio [OR], 1.63; 95% CI, 1.11 to 2.38; P=0.013). When we included the same covariates but did not adjust for differences in population admixture, the corin variant group remained independently associated with higher risk for prevalent hypertension (OR, 1.57; 95% CI, 1.08 to 2.29; P=0.018).
We then analyzed the data after excluding the 6 individuals who were homozygous for the minor corin allele (3 of 6, 50%, had hypertension). Multivariate logistic regression analysis that included adjustment for population admixture demonstrated that the corin variant group (all heterozygous individuals for the minor corin allele) remained independently associated with a higher risk for prevalent hypertension (OR, 1.68; 95% CI, 1.14 to 2.48; P=0.009). When we analyzed the entire black cohort, including the subjects <35 years of age, the corin variant group demonstrated an increased risk for prevalent hypertension (OR, 1.51; 95% CI, 1.06 to 2.15; P=0.022) in multivariate analysis using the same covariates as above.
We next analyzed the association of the corin T555I/Q568P allele with continuous measures of systolic, diastolic, and mean arterial blood pressures in untreated black DHS participants. We defined "untreated" as the black participants with no self-reported use of any type of antihypertensive medication based on the visit 1 survey. As demonstrated in Table 4, the corin variant group compared with the nonvariant group had significantly higher systolic and mean arterial blood pressures and a strong trend for a higher mean diastolic blood pressure in unadjusted analysis. In analysis that included an adjustment for differences in estimated population admixture and other potential confounders, the corin variant group had higher systolic, mean arterial, and diastolic blood pressures compared with the nonvariant group.
Association of Minor Corin Allele With Prevalent Hypertension and Blood Pressure in the MESA
We attempted to validate these associations in MESA. MESA was made up of an older population of blacks compared with DHS (Table 2), and the overall prevalence of hypertension was greater. As in DHS, the corin variant group had a higher prevalence of hypertension compared with the nonvariant group (Figure 2). In multivariate logistic regression (Table 5) analysis that included adjustment for age, gender, body mass index, and prevalent diabetes, the presence of the I555 (P568) allele remained independently associated with a higher risk for hypertension in MESA. However, unlike the DHS, when we analyzed the untreated black participants, there were no significant differences in the mean systolic, diastolic, or mean arterial blood pressures in the corin variant and nonvariant groups. Of note, more subjects in the corin variant group (114 of 208, 55%) than in the nonvariant group (728 of 1531, 48%) were being treated with antihypertensive medication (P=0.049) and thus had to be excluded from analysis.
Assessment of Dose-Response Effect of Minor Corin Allele
We combined the black participants in the DHS and MESA to examine a genotype-phenotype "dose-response" relationship given the small number of homozygous participants in each of the 2 individual cohorts. There was the suggestion of an increase in the prevalence of hypertension across the genotype groups in the combined cohort: participants homozygous for major allele (1454 of 2789, 52%), heterozygous for minor corin allele (228 of 380, 60%), and homozygous for minor allele (10 of 15, 66%) (P=0.002 for trend). However, the small number of homozygous subjects weakens the strength of this conclusion.
Association of Minor Corin Allele With Blood Pressure in the Chicago Genetics of Hypertension Study
The Chicago Genetics of Hypertension Study provided the opportunity to validate the association of the corin I555 (P568) allele with continuous blood pressure parameters because by design all participants were not using antihypertensive medication. The Chicago Genetics of Hypertension Study included a population with an age range (Table 2) similar to that of the black participants in the DHS. Unlike the DHS and MESA, this study excluded participants with a self-reported prior diagnosis of hypertension or the current use of an antihypertensive medication, creating the potential to bias against finding an association of the minor corin allele with higher blood pressure. Nonetheless, despite this potential, the corin variant group demonstrated significantly higher systolic and mean arterial blood pressures (Table 6) and a similar trend for higher diastolic blood pressure. These results persisted in adjusted analysis.
The data herein describe a minor corin I555 (P568) allele characterized by the presence of 2 nonsynonymous, nonconservative (type 1) SNPs that are enriched in blacks and associated with a higher risk for hypertension and higher blood pressure in untreated individuals. The increasing number of recognized signaling pathways and molecules involved in mammalian blood pressure regulation, small biological effects attributable to single polymorphisms, and conceptual and methodological study limitations have provided significant challenges to dissect the genetic contributions to interindividual variability in blood pressure and risk for the development of hypertension. Lack of reproducibility of genetic association studies for complex traits has created concern for false-positive associations and prompted prominent editorial boards to recommend that investigators seek replication of positive associations in independent population samples.34
Given these concerns, we believe that the replication of the results demonstrated in our primary cohort, the Dallas Heart Study, in 2 additional, independent population-based cohorts (MESA and Chicago Genetics of Hypertension Study) is a major strength of the present analysis. Such replication reduces the risk for a false-positive association caused by multiple testing. In addition, in our primary cohort (DHS), we were able to control for population stratification, another major cause of false-positive gene association studies, using 2 validated statistical methods employing 2114 highly informative SNPs.24 The corin variant group demonstrated a small but statistically significant greater estimate of African ancestry. However, adjusting our data for population stratification by 2 different methods did not reduce the association of the minor corin allele with a higher risk for prevalent hypertension or the association of the minor corin allele with higher blood pressure. Our approach was based on an analysis of SNPs that were prioritized on the basis of several characteristics described earlier. Such approaches require testing multiple SNPs for association and raises concerns about multiple testing. However, replication of the association to various degrees in 2 independent cohorts reduces the possibility of a type I error. Another approach that we did not use that may increase statistical power and reduce the problem of multiple testing is to incorporate a haplotype-based approach across the candidate gene region.
Corin is a plausible candidate gene for association with hypertension on the basis of the recognized importance of the natriuretic peptide system to blood pressure regulation.35–38 At the most proximate limb of the natriuretic peptide system, corin is required to process natriuretic peptide precursors effectively into smaller biologically active molecules. Impairment of this process might contribute to altered blood pressure regulation, as suggested recently from the report that homologous deletion of the corin gene in mice results in higher blood pressure and cardiac hypertrophy in the corin gene knockout mice.39 Additionally, the minor corin allele is defined by the presence of 2 missense SNPs, and the amino acid changes are nonconservative, occur in an important functional domain, and demonstrate strong conservation among species. Finally, the magnitude of the increased odds for prevalent hypertension in the corin variant group is consistent with the expected magnitude for a single gene contribution to complex phenotypes, as demonstrated by previous gene association studies for complex traits that have been successfully replicated.40
Our 3 different cohorts provided complementary strengths to determine the association of the minor corin allele with the risk for higher blood pressure and hypertension. The DHS and Chicago Genetics of Hypertension trials recruited large numbers of younger untreated black subjects, thereby providing the opportunity to test for linear associations with blood pressure levels when analyzed as continuous variables. In contrast, MESA provided the opportunity to confirm the hypothesized association between corin genotype and prevalent hypertension in a significantly older population in which hypertension was very common and antihypertensive treatment had already been initiated in a substantial proportion of participants. However, the greater prevalence of treated hypertension in MESA likely contributed to our inability to replicate the association of the corin minor allele with blood pressure levels in untreated subjects.
We recognize that in the absence of functional studies, we cannot conclude that the association of the minor corin T555I (Q568P) allele with higher blood pressure and risk for prevalent hypertension was mediated by a reduction in the ability of corin to process pronatriuretic peptides into their biologically active moieties. Rather, T555I and Q568P may be in linkage disequilibrium with another unrecognized causal variant. It is intriguing, however, to hypothesize that T555I and/or Q568P may alter the bioactivity of corin. These polymorphisms result in nonconservative amino acid substitutions within the second cysteine-rich frizzled-like domain in exon 12 of corin. The cysteine-rich frizzled domains are important for Wnt-signaling pathways, protein-protein interactions,41,42 and adequate catalytic activity of corin. Deletion of the first cysteine-rich frizzled domain reduced the ability of corin to process pro-ANP by 40%.33 Similarly, deletion of the second cysteine-rich frizzled-like domain, in which T555I and Q568P are located, reduced the catalytic function of corin by 50% to 60% (Q.W., Berlex Biosciences, personal communication).
The present study has limitations. Using a candidate gene approach, we have tested multiple SNPs for association with hypertension, which raises concerns about an increased risk of false-positive associations. To reduce this risk, however, we were committed a priori to replication of initial associations in additional independent cohorts. We were unable to replicate the association of the corin genotype with untreated blood pressure levels in MESA, but we reason that this is explained by the small number of untreated hypertensive participants in MESA and the exclusion of persons with known cardiovascular disease, which may have biased against our phenotype of interest. The small number of subjects homozygous for the minor I555 (P568) allele limited our ability to test a genotype-phenotype dose response across the 3 genotype groups.
It should be acknowledged that despite the demonstrated higher relative odds for prevalent hypertension associated with the minor corin T555I (Q568P) allele, the attributable risk for hypertension associated with the corin T555I/Q568P allele will be modest (&2% to 3%) and cannot explain the excess hypertension in US blacks relative to whites. The potential exists that overemphasis on genetics as a major explanatory factor for health disparities may result in researchers overlooking other equally important factors contributing to these disparities and may inadvertently reinforce racial stereotyping, as recently pointed out by others.43
In conclusion, the present data demonstrate that the corin I555 (P568) allele was enriched in blacks and was associated with higher blood pressure and a higher risk for prevalent hypertension.
Drs Dries, Victor, Leonard, Cooper, Rame, and Drazner received support from the Donald W. Reynolds Cardiovascular Clinical Research Center, Dallas, Tex. Drs Dries and Drazner were supported as Reynolds Associates. In addition, Dr Dries received support from the NIH, NHLBI (K23-HL04455). Dr Drazner was the recipient of a Doris Duke Clinical Scientist Development Award from the Doris Duke Charitable Foundation, New York, NY. Dr Cooper received support from the NIH, NHLBI (RO-1 45508; 47910). Drs Cooper and Post are investigators for the NHLBI-funded MESA. Dr Post is a Paul Beeson Physician Faculty Scholars in Aging Research Award recipient and a Reynolds Associate.
Dr Wu is consultant for and former employee of Berlex Biosciences.
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- Corin Gene Minor Allele Defined by 2 Missense Mutations Is Common in Blacks and Associated With High Blood Pressure and HypertensionDaniel L. Dries, Ronald G. Victor, J. Eduardo Rame, Richard S. Cooper, Xiaodong Wu, Xiaofeng Zhu, David Leonard, Su-Inn Ho, Qingyu Wu, Wendy Post and Mark H. DraznerCirculation. 2005;112:2403-2410, originally published October 17, 2005http://dx.doi.org/10.1161/CIRCULATIONAHA.105.568881
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- Corin Gene Minor Allele Defined by 2 Missense Mutations Is Common in Blacks and Associated With High Blood Pressure and HypertensionDaniel L. Dries, Ronald G. Victor, J. Eduardo Rame, Richard S. Cooper, Xiaodong Wu, Xiaofeng Zhu, David Leonard, Su-Inn Ho, Qingyu Wu, Wendy Post and Mark H. DraznerCirculation. 2005;112:2403-2410, originally published October 17, 2005http://dx.doi.org/10.1161/CIRCULATIONAHA.105.568881
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Investors eye data and banks' moves
FOLLOWING a week of thin trading over the Christmas and New Year holiday period, investors are set to get down to business this week.
They are expected to focus on manufacturing data from Singapore and Hong Kong, as well as on China trade data for indications on the health of the world's second-largest economy.
The strength of Asia's manufacturing economy - a key component of economic strength - will be in focus this week with Singapore today posting its Purchasing Managers' Index (PMI) for last month, and Hong Kong releasing its PMI tomorrow.
The two reports follow survey results last week showing weak factory activity in China last month, as the country's manufacturers fight rising costs and softening demand.
China's official PMI slipped to 50.1 last month from November's 50.3, with weaker-than-expected oil-demand growth in China last year contributing to the price collapse.
"Expectations are for Singapore's manufacturing activity to be weak because of the slowdown in GDP (gross domestic product) growth, and high labour costs driving up costs for manufacturers. The United States and euro zone haven't recovered fast enough to help our exports," remisier Alvin Yong said.
Investors are also eyeing China trade figures for last month, with analysts forecasting China's foreign trade surplus may have narrowed with growth in exports outweighed by a sharper slide in imports. Indeed, some expect that China's economic growth for last year may undershoot the government's 7.5 per cent target, marking the weakest expansion in more than two decades.
"But more bad news from China and the euro zone could boost expectations of more stimulus, which is good for our local market," Mr Yong said. "We are hoping for a rally here in the first three to five working days, as that could set the tone for the trading year ahead."
The benchmark Straits Times Index ended the week 16.91 points, or 0.5 per cent, higher at 3,370.59.
On Wednesday, the US Federal Reserve is expected to release minutes of the Federal Open Market Committee meeting on Dec 16 and 17, which may shed light on the timing of the first interest rate increase since 2006. Last month, the committee had said it would be "patient" on the timing of an increase, replacing an earlier pledge to keep borrowing costs low for a "considerable time".
More indications on what the Fed intends to do about interest rates will come from the US employment report for last month, out on Friday.
A Bloomberg survey as of Dec 30 showed that the jobless rate probably declined last month to a more than six-year low, as employers added almost 250,000 jobs during the month to cap the strongest year for payroll growth since 1999.
On Thursday, the Bank of England's Monetary Policy Committee is expected to announce a decision on whether to raise the benchmark interest rate from a record-low 0.5 per cent.
While two officials have voted in recent months to raise borrowing costs, citing the potential for pay pressures to build, most economists do not see a rate increase until the third quarter this year.
A report last week showed that British manufacturing growth unexpectedly slowed last month, reducing pressure on the Bank of England to raise interest rates.

subset: commoncrawl


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ration sth out
(English pronunciations of "ration sth out" from the Cambridge Advanced Learner's Dictionary & Thesaurus and from the Cambridge Academic Content Dictionary, both sources © Cambridge University Press)
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Welcome to Librarium Online!
Join our community of 80,000+ members and take part in the number one resource for Warhammer and Warhammer 40K discussion!
Registering gives you full access to take part in discussions, upload pictures, contact other members and search everything!
Hello Chaos Space Marine players!
The old (and, unfortunately, current) Codex: Daemonhunters features a scenario called Stop the Ritual to be played against Chaos Space Marines.
I have revised it for fifth edition. And since I like to play scenarios written by third parties (so that both players have the same preconditions), I also like to share my scenarios for others to play. Maybe some of you will be grateful for a non-standard but (hopefully) balanced mission with a fluffy backstory. Please note that I am not a Chaos Space Marine player, and also that the scenario can be played with any army as an attacker (and also with any army as a defender, if you want). Enjoy!
What has been changed:
Here are the rules...
- Fifth edition does not use sentries. Thus, the defender (Chaos Space Marines) may now act normally right from the beginning.
- Since 5+ cover saves are rather weak in fifth edition, the models incanting the ritual instead receive a 4+ cover save.
- To prevent devastating alpha-strikes, all of the attacker's vehicles must begin the game in reserves.
- And most importantly: I thought it was a little mean that the Chaos Space Marine player is forced to designate his summoning circles at the beginning of the game and that those units could not act throughout the entire game at all. Thus in this version, any infantry unit of at least five models (that meets certain requirements as outlined in the rules below) can form a summoning circle at any time during the game. And as long as there is always at least one intact summoning circle at the end of every Chaos turn (and at the end of the game), the Chaos Space Marines win.
The Chaos Marines are attempting to open a rift into the Warp and release all hell. A brave fighting force arrives to attempt to stop them last minute.
The Set-Up
The Chaos Marines deploy within 18" of the table center (preferably marked by a ruined temple or altar). The opposing force then picks a short table edge as its own. The opposing force deploys within 6" of its table edge. The opposite short table edge is the Chaos Marines'.
Any vehicles in the attacker's army must start the game in reserves.
Then both forces deploy infiltrators and conduct scout moves as usual.
Roll off to see who goes first: whoever scores highest has the choice. Initiative can not be stolen: whoever has been chosen to go first immediately starts the game with their first turn.
Game length is the same as for standard missions. The mission has the special rules Reserves and Deep-Strike. Note that the rules for outflanking are unaltered, any outflanking units will still arrive from the short table edges.
Mission Mechanics
The Chaos Marines must continue to incant the summoning ritual. This must be done by a summoning circle. Any unit of at least five models, which is arranged in a closed circle with no more than 2" between any two neighboring models enclosing the center of the board, and which is not falling back or has gone to the ground is considered a summoning circle.
A summoning circle may not act other than move to attain or retain their closed circular formation, react to an assault, or fall back. Models in a summoning circle are protected by a 4+ cover save from the coruscating energies swirling around them. A summoning circle which has fewer than five models, has more than 2" between any two of its neighboring models, does not enclose the center of the table, is falling back or has gone to the ground does not count as incanting the summoning ritual.
If either at the end of any Chaos Marine turn or the end of the game no unit is incanting the summoning ritual, the attackers win. If at the end of every Chaos Marine turn and the end of the game there is a unit incanting the summoning ritual, the Chaos Marines win.
Any feedback welcome!
Red Archer's BLOG
Well done mister Archer.
The Wolf time is upon us!
Rep for the Rep God! Positive for the Positive Throne!
Armies: Space Wolves, Imperial Guard, Emperors Children, Necrons, Eldar


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HPE opens up over how customers can utilise the Azure cloud in their own data centre with HP kit
German Regulator Orders WhatsApp To Halt Data Harvest
Breaking the law…Watchdog accuses WhatsApp of 'misleading' users and breaking German data protection laws
Microsoft Signs Connected Car Deal With Renault-Nissan
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Yes folks, Apple did make some technology turkeys. The Apple Newton PDA being a case in point


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This study examined the transduction pathways activated by epinepherine in the pacemaker region of the toad heart. Recordings of membrane potential, force, and intracellular Ca2+ concentration ([Ca2+]i) were made from arrested toad sinus venosus. Sympathetic nerve stimulation activated non-α-, non-β-adrenoceptors to evoke a membrane depolarization and a transient increase in [Ca2+]i. In contrast, the β-adrenoceptor agonist isoprenaline (10 μM) caused membrane hyperpolarization and decreased [Ca2+]i. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.5 mM) mimicked the isoprenaline-evoked membrane hyperpolarization. Epinephrine (10–50 μM) caused an initial membrane depolarization and an increase in [Ca2+]i followed by membrane hyperpolarization and decreased [Ca2+]i. The membrane depolarizations evoked by sympathetic nerve stimulation or epinephrine were abolished either by the phospholipase C inhibitor U-73122 (20 μM) or by the blocker ofd-myo-inositol 1,4,5,-trisphosphate-induced Ca2+ release, 2-aminoethoxydiphenyl borate (2-APB, 60 μM). Neither U-73122 nor 2-APB had an affect on the membrane hyperpolarization evoked by β-adrenoceptor activation. These results suggest that in the toad sinus venosus, two distinct transduction pathways can be activated by epinephrine to cause an increase in heart rate.
- cardiac
- inositol trisphosphate
- cAMP
stimulation of the sympathetic nerves innervating mammalian and amphibian hearts causes an increase in heartbeat rate (12, 21). These responses are generally assumed to result from the activation of postjunctional β-adrenoceptors by catecholamines, with such β-adrenoceptors being linked to the activation of adenylate cyclase and production of cAMP (13). However, catecholamines released from sympathetic nerves innervating amphibian and mammalian cardiac pacemaker tissue appear to combine with a distinct set of adrenoceptors linked to a pathway that does not involve cAMP (4,6, 8). In both mammals and amphibians, the adrenoceptors linked to the cAMP-independent pathway are likely to have a junctional location residing within the neuroeffector cleft and are stimulated readily by a neurally released transmitter. Exogenously applied transmitters do not readily activate junctional receptors but easily activate extrajunctional receptors that are located outside the neuroeffector cleft and are linked to the cAMP-dependent pathway (4, 6, 8). A comparison of the differences between neurally released and exogenously applied catecholamine is most easily studied in amphibians where, in contrast to mammals, the junctional and extrajunctional adrenoceptors are pharmacologically distinct, junctional receptors being non-α-, non-β-adrenoceptors and extrajunctional receptors being β-adrenoceptors. In amphibians, the differences between the activation of the two types of adrenoceptors are most apparent in pacemaker preparations in which the discharge of pacemaker action potentials has been prevented by organic Ca2+ antagonists. In these arrested preparations, the activation of β-adrenoceptors causes membrane hyperpolarization as does the elevation of cAMP. In contrast, sympathetic nerve stimulation initiates a membrane depolarization that does not appear to involve the elevation of cAMP (4). The adrenoceptors activated either by a neuronally released transmitter or high concentrations of rapidly applied epinephrine cannot be blocked with either α- or β-adrenoceptor antagonists (6, 19). However, such receptors can be blocked with dihydroergotamine (6). This indicates that both neuronally released epinephrine and bath-applied epinephrine can activate a set of dihydroergotamine-sensitive receptors that are non-α-, non-β-adrenoceptors. When activated, these receptors cause the release of Ca2+ from intracellular stores and initiate positive chronotropic and inotropic responses (5). Furthermore, transient increases in intracellular Ca2+ concentration ([Ca2+]i) accompany membrane depolarization evoked by stimulation of the junctional non-α-, non-β-adrenoceptors in arrested preparations (9). In many other preparations, transmitter-evoked release of Ca2+ from intracellular Ca2+ stores is triggered by the production of d-myo-inositol 1,4,5,-trisphosphate [Ins(1,4,5)P3] (2). First, this study examined the differences between the responses produced by β-adrenoceptor activation and those produced by sympathetic nerve stimulation and the activation of non-α-, non-β-adrenoceptors in arrested preparations of toad sinus venosus. Second, the hypothesis was tested that, in the toad sinus venosus, the stimulation of non-α-, non-β-adrenoceptors results in the activation of a pathway involving the elevation of Ins(1,4,5)P3.
The procedures described have been approved by the animal experimentation committee at the University of Melbourne. Toads (Bufo marinus) were anesthetized by immersion in a solution of 0.5% tricaine methanesulfonate (Thomson and Joseph, Norwich, UK) in tap water. Preparations consisted of the sinus venosus and two atria, with the ventricle and truncus arteriosus removed. Both the left and right vagosympathetic trunks were dissected free back to the sympathetic chains and left intact with the sinus venosus. Preparations were pinned flat in a shallow recording chamber, the base of which consisted of a microscope coverslip coated with Sylgard silicone resin (9) and continuously perfused with physiological saline solution composed of (in mM) 115 NaCl, 3.2 KCl, 20 NaHCO3, 3.1 NaH2PO4, 1.8 CaCl2, 1.4 MgCl2, and 16.7 glucose gassed with 95% O2-5% CO2 at a rate of 6 ml/min (bath volume, 1.5 ml) unless otherwise stated. In some experiments, force generated by the tissue was also measured by placing a fine platinum hook attached to a tension transducer through the sinus venosus. The discharge of pacemaker action potentials was prevented by adding nifedipine (10 μM) to the perfusion fluid (6). Experiments were carried out at room temperature (22–25°C). The sympathetic nerves were stimulated by passing the sympathetic trunks through a pair of platinum ring electrodes (stimulation currents, 10–60 nA; pulse width, 1.0 ms). Sympathetic nerves were stimulated at 10 Hz for 1 s. Pilot experiments using long trains of sympathetic nerve stimulation revealed that a 1-s train of sympathetic stimuli is maximal. That is, there is no change in the amplitude, duration, or complexity of response with increasing the duration of sympathetic stimuli (up to 30 s). Drugs were added to the preparation by changing the inflow line from the control solution to one containing the appropriate concentration of drug. When epinephrine and isoprenaline were perfused onto the sinus venosus, the perfusion rate was increased to 12 ml/min. Membrane potential recordings were made using conventional techniques with fine glass microelectrodes (resistance, 120–240 MΩ) filled with 0.5 M KCl.
In some experiments, concurrent measurements of membrane potential and relative changes in [Ca2+]i were made from pinned sinus venosus preparations loaded with the fluorescent Ca2+ indicator fura 2. This was achieved by incubation of the sinus venosus preparations in HEPES-buffered physiological saline solution containing the membrane permeable form of fura 2, fura 2-acetoxymethyl ester (fura 2-AM, 10 μM), and a low concentration of Ca2+ (0.1 mM). The dispersal of fura 2-AM into the muscle cells was aided by the addition of Pluronic F127 (0.01%) to the buffer solution. After incubation for 2.5 h at 22°C, preparations were then warmed to 32°C for 30 min before washing in fura 2-AM-free physiological saline solution for 40 min. The ratio of the fluorescence recorded at 510 nm after excitation with 340-nm light to that recorded after excitation with 380-nm light (F340/380) was taken as a qualitative indicator of [Ca2+]i.
All values are expressed as means ± 1 SE. Each n value represents the mean result from a different animal. Where indicated, the statistical significance of the difference between two means was determined using a Student's t-test.
Drugs used were fura 2-AM (Molecular Probes; Eugene, OR), epinephrine bitartrate, dihydroergotamine tartrate, isoprenaline hydrochloride, nifedipine hydrochloride, propranolol hydrochloride, 3-isobutyl-1-methylxanthine (IBMX) (Sigma; St. Louis, MO), U-73343, U-73122 (Sapphire Bioscience; New South Wales, Australia), and 2-aminoethoxydiphenyl borate (2-APB, Calbiochem; Croydon, Victoria, Australia). All drugs were dissolved in distilled water except nifedipine and 2-APB, which were dissolved in absolute alcohol, and fura 2-AM, U-73343, and U-73122, which were dissolved in dimethyl sulfoxide (ICN Biomedicals; Aurora, OH). In all experiments, solutions containing nifedipine were made daily and protected from light using aluminum foil wrapping.
Effects of isoprenaline, epinephrine, and sympathetic nerve stimulation on membrane potential and [Ca2+]i in the arrested sinus venosus.
Membrane potential recordings were made from the sinus venosus, the pacemaker region of the toad heart. In the presence of nifedipine (10 μM), the membrane potential was stable and lay in the range of −31 to −39 mV, mean −35 ± 1 mV (n = 25 hearts).
In arrested preparations of toad sinus venosus, trains of sympathetic nerve stimuli (10 impulses at 10 Hz) caused a complex membrane depolarization that typically displayed oscillations (Fig.1 A, trace a) (4, 6). These depolarizations had amplitudes of 25.2 ± 2.1 mV (n = 7) and were initiated after a latency (time elapsed between the start of the stimulation train and the attainment of 10% of the peak amplitude) of 1.27 ± 0.04 s. When the [Ca2+]i was monitored simultaneously, it was apparent that the depolarizations produced by sympathetic nerve stimulation were associated with an increase in [Ca2+]i (Fig. 1 A, trace b) (5). Stimulation with 10 impulses delivered at 10 Hz evoked an increase in [Ca2+]i with an amplitude of 0.15 ± 0.03 F340/380 (n= 5).
In contrast, in arrested preparations, the activation of β-adrenoceptors evoked a membrane hyperpolarization (Fig.1 B, trace a) (4). Rapid perfusion of isoprenaline (50 μM) over the sinus venosus for 30 s evoked a membrane hyperpolarization that had a mean nadir of 3.5 ± 0.1 mV and lasted ∼5 min (n = 3). This membrane hyperpolarization was associated with a decrease in [Ca2+]i of 0.12 ± 0.02 F340/380 (Fig. 1 B, trace b;n = 3) (10). This hyperpolarization was mimicked by activating adenylate cyclase or by inhibiting phosphodiesterases (4). Thus the addition of the phosphodiesterase inhibitor IBMX (0.5 mM) to the solution bathing the sinus venosus evoked a membrane hyperpolarization with a similar magnitude to that evoked by β-adrenoceptor activation (Fig.1 C). The ionic basis of the membrane potential change produced by isoprenaline or by IBMX detected in arrested amphibian pacemaker cells remains unclear. However, it is clear that β-adrenoceptor agonists and agents that cause the elevation of cAMP produce similar sequences of membrane potential changes (4,13) but ones quite different to those produced by sympathetic nerve stimulation.
The rapid perfusion of epinephrine (10 μM) onto the arrested sinus venosus evoked an initial transient membrane depolarization of 9.7 ± 1.2 mV followed by a hyperpolarization with peak nadir of 3.3 ± 0.6 mV (n = 10; Fig.2, A, trace a, andB, trace a). The depolarization was associated with an increase in [Ca2+]i, whereas hyperpolarization was associated with a fall in [Ca2+]i (Fig. 2 A, trace b, n = 5) (10). On some occasions, membrane potential oscillations were superimposed on the falling phase of the depolarization (Fig. 5 B, trace a). These oscillations had a similar frequency to those evoked by sympathetic nerve stimulation. The membrane hyperpolarization, which resembled that produced by either isoprenaline or IBMX, was abolished by pretreating the tissues with the β-adrenoceptor antagonist propranolol (1 μM; Fig. 2 B, trace b) suggesting that it resulted from β-adrenoceptor activation. In the presence of propranolol (1 μM), applied epinephrine evoked only a depolarization that was abolished by dihydroergotamine (10 μM; Fig. 2 B,trace c) (6). This effect of dihydroergotamine was not due to its action as an α-adrenoceptor antagonist because membrane depolarizations persist in the presence of other α-adrenoceptor antagonists (6). These results suggest that the rapid bath application of epinephrine results in the activation of non-α-, non-β-adrenoceptors like that produced by sympathetic nerve stimulation as well as the activation of β-adrenoceptors (4, 6, 9).
Effect of phospholipase C inhibition on responses to epinephrine and sympathetic nerve stimulation in arrested sinus venosus.
Previous experiments show that sympathetic nerve stimulation and applied isoprenaline initiate distinct sequences of membrane potential changes presumably by activating different metabolic pathways. Sympathetic nerve stimulation is associated with an increase in [Ca2+]i resulting from Ca2+ store release that does not involve the activation of cAMP (4,9). Very often Ca2+ store release results from the activation of an Ins(1,4,5)P3-dependent pathway (2). The possibility that such a pathway mediated responses resulting from the activation of non-α-, non-β-adrenoceptors was investigated by examining the effects of inhibiting phospholipase C (PLC) with U-73122 (3,23) on responses evoked by sympathetic nerve stimulation. Responses evoked by sympathetic nerve stimulation (10 impulses at 10 Hz, n = 5) were abolished with U-73122 (20 μM). However, the effect of this agent may have resulted in part, if not entirely, from effects on transmitter secretion. In parallel sets of experiments, U-73122 (20 μM) abolished the responses to vagus nerve stimulation (n = 3). To avoid this complication, non-α-, non-β-adrenoceptors were activated by the rapid application of epinephrine. Figure 3 A,traces a and b, shows that the PLC inhibitor U-73122 (20 μM) abolished the membrane depolarization produced by applied epinephrine (n = 5). However, the membrane hyperpolarization resulting from the activation of β-adrenoceptors was unchanged.
This indicates that U-73122 neither blocks β-adrenoceptors nor interferes with the cAMP-dependent pathway. The simplest explanation is that U-73122 inhibits the depolarization evoked by the activation of non-α-, non-β-adrenoceptors by preventing PLC activation. As a further control, the effects of the closely related compound U-73343 (3) on the responses to applied epinephrine were examined. U-73343 (20 μM), which does not block PLC, had no effect on the amplitude of either the depolarization or the afterhyperpolarization produced by added epinephrine (P > 0.05,n = 5; Fig. 3 B, traces a andb).
In a number of tissues, it has been shown that, in addition to inhibiting PLC, U-73122 may also cause the release of Ca2+from intracellular stores (16, 18). To examine whether the effects of U-73122 were due to Ca2+ store depletion, the effect of U-73122 on responses evoked by application of caffeine (10 mM) was examined. It has previously been shown (5) that the rapid bath application of high concentrations of caffeine causes a membrane depolarization that is oscillatory in nature (Fig.4 A, trace a). This is associated with an oscillatory increase in force production of the sinus venosus (Fig. 4 A, trace b). These changes in membrane potential and force are due to the release of Ca2+ from ryanodine-sensitive stores (5). U-73122 (20 μM) had little effect on the underlying membrane depolarization or increase in force production evoked by caffeine; however, it abolished the oscillations in both membrane potential and force (Fig. 4 B). Therefore, part of the effects of U-73122 on responses evoked by sympathetic nerve stimulation may have been due to its action on Ca2+ stores rather than PLC inhibition.
To further examine the possibility that Ins(1,4,5)P3production was necessary for the response produced by the activation of non-α-, non-β-adrenoceptors, the effects of inhibition of Ins(1,4,5)P3-induced Ca2+ release by 2-APB (60 μM) (1, 17) were examined on both membrane potential and force changes evoked by sympathetic nerve stimulation. In control solution, stimulation of the sympathetic nerves evoked a membrane depolarization of 19 ± 5 mV (n = 5). The addition of 2-APB (60 μM) to the physiological saline solution (30 min) had no effect on either the resting membrane potential or the basal force production. However, 2-APB significantly reduced the amplitude of the nerve-evoked membrane depolarization (4 ± 2 mV, P= 0.01, n = 5, Fig.5 A). Washout of 2-APB for 60 min partially restored the response to sympathetic nerve stimulation (15 ± 3 mV). In four experiments, the effect of 2-APB (60 μM) on force changes evoked by sympathetic nerve stimulation was also examined. Before the addition of 2-APB, sympathetic nerve stimulation evoked an increase in basal force of 0.28 ± 0.10 mN. After the addition of 2-APB, the increase in force production evoked by sympathetic nerve stimulation was significantly reduced (0.03 ± 0.01 mN; P = 0.02). At 60 μM, 2-APB had no effect on responses evoked by vagus nerve stimulation (n = 3), suggesting that it was not inhibiting transmitter release.
The effect of 2-APB was also examined on responses evoked by the bath application of epinephrine. If 2-APB was acting specifically to inhibit Ins(1,4,5)P3-induced Ca2+ release, it would be expected that 2-APB would abolish the membrane depolarization resulting from the activation of non-α-, non-β-adrenoceptors but have no effect on the membrane hyperpolarization resulting from β-adrenoceptor activation. Epinephrine (50 μM) evoked an initial membrane depolarization of 7 ± 2 mV followed by a membrane hyperpolarization of 4 ± 1 mV (n = 4) in amplitude (Fig. 5 B, trace a). The addition of 2-APB (60 μM, 30 min) significantly reduced the amplitude of the membrane depolarization (1 ± 1 mV; P = 0.04) but had no effect on the membrane hyperpolarization (5 ± 1 mV;P = 0.09; n = 4; Fig. 5 B,trace b).
Although 2-APB (60 μM) has previously been shown to have no effect on either the production of cAMP or Ca2+-induced Ca2+ release, 2-APB has been shown to partially inhibit the reuptake of Ca2+ into Ins(1,4,5)P3-sensitive Ca2+ stores when used at high concentrations (≥100 μM) (17). To examine whether 2-APB was depleting intracellular stores due to the inhibition of Ca2+reuptake, the effect of 2-APB on changes in membrane potential and force evoked by caffeine was examined. Both the membrane depolarization and increase in force evoked by caffeine persisted in the presence of 2-APB (60 μM, n = 6, Fig.6). In addition, both the oscillations in membrane potential and force production also persisted in the presence of 2-APB.
This study suggests that two distinct transduction pathways, one involving cAMP and the other involving Ins(1,4,5)P3, are present and can be activated by catecholamines in the toad sinus venosus. In the toad sinus venosus, just as in mammalian pacemaker preparations, applied catecholamines most readily activate β-adrenoceptors to elevate cAMP. This pathway results in the phosphorylation-dependent and -independent modulation of voltage-dependent channels involved in pacemaking activity (11,13). The resulting tachycardia is associated with an increase in the amplitude of both pacemaker action potentials (4, 6) and the [Ca2+]i transient that accompanies each action potential (5).
In contrast, responses evoked by sympathetic nerve stimulation resulting from activation of non-α-, non-β-adrenoceptors are not associated with an increased influx of Ca2+ through voltage-dependent Ca2+ channels (5) and do not involve cAMP (4). However, such responses are likely to involve a different complex metabotropic pathway; both the sympathetic tachycardias recorded from beating preparations and the membrane depolarizations recorded in arrested preparations begin after a long latency and last many seconds even when triggered by only a few sympathetic stimuli (4, 9). In arrested preparations, stimulation of the sympathetic nerves evokes an oscillatory membrane depolarization and a concomitant increase in [Ca2+]i (9), both of which have temporal characteristics similar to the sympathetic tachycardia recorded from beating preparations (6). This suggests that a similar transduction pathway is activated in beating and arrested preparations of toad sinus venosus. We have recently shown that both the membrane depolarization and the increase in [Ca2+]i evoked by sympathetic nerve stimulation in arrested preparations result from the release of intracellularly stored Ca2+ (9). Similar responses also resulting from the activation of non-α-, non-β-adrenoceptors can be produced by rapid bath application of epinephrine to the arrested pacemaker tissue (Fig. 2) (4,6). Responses evoked by the activation of these receptors were inhibited by prevention of Ins(1,4,5)P3-induced Ca2+ release. This was achieved by either preventing the formation of Ins(1,4,5)P3 with the PLC inhibitor U-73122 or by directly preventing Ins(1,4,5)P3-induced Ca2+ release with 2-APB (Figs. 3 and 5). This suggests that the responses involve the activation of PLC and the formation of Ins(1,4,5)P3, which triggers the release of Ca2+ from intracellular stores. The subsequent activation of the Na+/Ca2+ exchanger found in cardiac muscle (7, 14) would provide a net inward current, which could account for both the depolarization in arrested preparations and the tachycardia in beating preparations (15).
How these findings relate to mammalian pacemaker cells remains unclear. In the mammalian heart, both neuronally released and added norepinephrine activate β-adrenoceptors. However, the responses to neuronally released and added norepinephrine differ in much the same way as they do in the amphibian heart (8). Clearly, one explanation could be that the β-adrenoceptors under sympathetic nerve terminals are linked to PLC, whereas those at a more distant location are linked to adenylate cyclase. However, we know of no example where a β-adrenoceptor is linked to PLC. Indeed in mammalian hearts, this pathway is usually linked to an α-adrenoceptor (20, 22).
Whatever the case, it is clear that in both amphibians and mammals, the same transmitter activates separate metabotropic pathways. One pathway is activated by the local high concentrations of transmitter achieved after nerve activity. In amphibians, this pathway appears to involve the second messenger Ins(1,4,5)P3, which releases Ca2+ from an intracellular store to increase heart rate. The other pathway, which involves cAMP, is activated by a more diffuse action of transmitter such as that occurring during a rise in the concentration of catecholamine circulating in the blood.
This work was funded by the National Health and Medical Research Council of Australia. We thank them for their financial support.
Address for reprint requests and other correspondence: N. J. Bramich, Dept. of Zoology, Univ. of Melbourne, Victoria, Australia 3010 (E-mail: narelleb{at}unimelb.edu.au).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2001 the American Physiological Society


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