Gonad recrudescence and annual sex steroid patterns in female goldspot mullet, Liza parsia reared in brackishwater pond.

Goldspot mullet, Liza parsia is a commercially important fish of South East Asia, where its farming depends on wild seed resources due to unavailability of hatchery technology. It, therefore, is important to understand the annual reproductive cycle of female L. parsia in captivity. In this study, adult male and female L. parsia (body weight ranges: 45-90 g; total length ranges: 100-125 mm, age >1 year) were collected from the wild and reared in a brackishwater pond. Thereafter, fish were randomly sampled at monthly intervals to measure the hepatosomatic index (HSI), gonadosomatic index (GSI), levels of serum steroids (testosterone, T; 17ß-estradiol, E2 and 17α-hydroxyprogesterone, 17-OHP), and oocyte growth. Results exhibited that female L. parsia undergoes six different maturation stages, namely I (oocyte diameter, OD: <100 µm), II (OD: 100-350 µm), III (OD: 350-400 µm), IV (vitellogenic oocyte, OD: 400-450 µm), V (ripe oocyte, OD: 450-550 µm) and VI (atretic oocyte, OD: 60-150 µm), with synchronous oocyte development. The highest (P < 0.05) HSI (1.96 ± 0.24) and GSI (12.01 ± 0.73) were recorded in December and January, respectively. Concentration of E2 gradually increased from August and reached its peak (807.67 ± 25.98 pg mL-1, P < 0.05) in December. The level of 17-OHP (85.87 ± 0.91 pg mL-1) was at its peak during the normal spawning month (January) (P < 0.05). Overall, the results indicated that L. parsia attains maturity in brackishwater pond, which is consistent with previous observations, and altogether provide the basis to develop a breeding technology in captivity through hormonal and environmental manipulations.

Nervous necrosis virus titration and antigen quantitation by indirect sandwich enzyme linked immunosorbent assay.

Viral nervous necrosis (VNN) is a serious disease of marine and brackishwater fishes caused by nervous necrosis virus (NNV) resulting in up to 100% mortality in early larval and juvenile stages. Adult fish when infected are asymptomatic and spread the virus vertically to the offspring through milt and eggs. Prevention of vertical transmission of the disease is by using disease free broodstock and vaccinating the brooders. Estimation of antigen content and virus titre is essential to determine antigen/virus concentration in VNN vaccine. A monoclonal antibody based indirect sandwich ELISA was developed to quantify the NNV antigen and to estimate the virus titre by TCID50 coupled ELISA. Mouse hybridoma clones secreting monoclonal antibodies (MAb) against the capsid protein of NNV was developed and characterised. The antibodies reacted specifically with the recombinant capsid protein and purified virus in western blot. Polyclonal antibodies against NNV were used as capture antibodies and MAbs were used as detection antibodies to optimise an indirect sandwich ELISA to detect and quantify capsid protein of NNV. The developed assay had a sensitivity of 390 ng/ml and could detect the virus in clinical samples. The assay coupled with TCID50 could be used to estimate the virus titre rather than by observing the CPE which is laborious and subjective.

Seasonal changes in the milt quality of spotted scat, Scatophagus argus: Implications for artificial propagation.

The current study investigated the correlation among sperm production (milt volume, spermatocrit and sperm count), sperm motility characters and biochemical composition of seminal plasma during different seasons to estimate the quality of semen in the Spotted scat (Scatophagus argus). The sperm of 120 males were collected over a period of 2 years seasonally viz., summer, pre-monsoon, monsoon and post-monsoon. During the collection period, it was observed that there were significant (p < 0.05) seasonal variations in the semen quality including sperm motility and production more in the summer season than other seasons. The osmolality of seminal plasma varied from 343.33 to 370.33 mOsm/kg. The pH of seminal plasma elevated from 7.37 to 7.63 during monsoon and summer seasons. The motility of S. argus sperms correlated with the concentration of biochemical compounds viz., Ca2+, Mg+ and Cl- during the summer season. Based on the present observation it could be highlighted that sodium and potassium were negatively correlated (p < 0.01 and p < 0.05) effect on total protein (r = -0.826; -0.956), glucose (r = -0.819; -0.717), cholesterol (r = -0.978; -0.849), AST (r = -0.853; -0.897), ALT (r = -0.970; -0.967), calcium (r = -0.850; -0.697), magnesium (r = -0.852; -0.843) and chloride (r = -0.906; -0.926). In conclusion, many physical and biochemical compositions of S. argus semen improved during that the summer and pre-monsoon season as compared to that of monsoon and post-monsoon. Fertility tests were performed to determine the ability of spermatozoa to fertilize an egg. Highest fertilization (79.92 ± 4.60%) and hatching (80.75 ± 3.89%) rates were observed during summer, while the rates were lowest during monsoon (40.50 ± 4.52%; 53.92 ± 7.38%). This work forms a compiled data for standard semen quality, which in turn will help to select good milters for hatchery production, cryopreservation and assisted breeding programs of this fish.

Non-lethal screening of Asian seabass (Lates calcarifer) by monoclonal antibody based indirect enzyme linked immunosorbent assay for viral nervous necrosis.

Viral nervous necrosis (VNN) is a serious viral disease of several species of farmed and wild fishes. Adult fish are asymptomatic and become carriers of the virus when infected with nervous necrosis virus (NNV) and they transmit the virus to the offspring through eggs. ELISA is ideal for non-lethal screening of adult fish for VNN. Asian seabass (Lates calcarifer) IgM was purified using Protein A affinity column and hybridoma clones secreting monoclonal antibodies (MAb) specific to the heavy chain of IgM was developed. An Indirect ELISA using anti-seabass IgM MAb was developed by optimizing all the reagents. The assay was used to screen adult Asian seabass from grow-out farms in comparison to RT-PCR. The assay was also used to assess the immune response in Asian seabass immunized with inactivated Red-spotted grouper NNV (RGNNV). Seabass IgM on SDS-PAGE analysis revealed three heavy chain bands of size 96, 82 and 76 kDa and a single light chain of size 25 kDa. Out of 18 positive hybridoma clones, two selected clones reacted specifically with the 76 kDa heavy chain band. Out of 28 serum samples of Asian seabass from grow-out farms 26 were positive for NNV antibodies while 22 were positive by RT-PCR. Fish immunized with inactivated RGNNV showed immune response by one week post-immunization, and the peak immune response was observed four weeks post-immunization. The assay developed can be used for non-lethal screening of adult Asian seabass for VNN and to assess the immune response after vaccination.

Gonadal recrudescence and annual reproductive hormone pattern of captive female Spotted Scats (Scatophagus argus).

The Spotted Scat (Scatophagus argus), an important euryhaline fish inhabiting mangrove and coastal regions of Indo-Pacific waters, is both an ornamental and food fish in India. Detailed insight into maturation of Spotted Scat when maintained in aquaculture systems, therefore, needs to be elucidated. Lack of information on annual maturation dynamics of female scat collected from their natural habitat and reared in earthen ponds is the basis of this study. Oocytes were classified into five developmental stages: pre-vitellogenic, vitellogenic, late-vitellogenic, ripe, and follicular atresia. Ovarian maturity stages were subsequently categorized as immature (Stage 1), vitellogenesis (Stage 2), maturing (Stage 3), mature (Stage 4), and spent (Stage 5). In oocytes in primary, secondary and tertiary yolk stages, there are greater concentrations of E2 in vitellogenic females between March and June. Significant increases of E2, T, and 17-OHP paralleled the increase of diameter of late-vitellogenic oocytes in maturing females during July. The completion of vitellogenesis and initiation of germinal vesicle migration in the cytoplasm were evident in mature females (Stage 4) with a decreasing trend of sex steroids in and subsequent to the month of August. There were 50 % of oocytes in the final oocyte maturation stage (FOM) (490-620 µm) until completion of Stage 4 in September. The results of this study indicate there is complete ovarian maturation in female scats captured in their natural habitat and maintained in an earthen pond, which may be important information for hatchery management for induction of spawning of Spotted Scat in aquaculture systems.

Ultrastructure of sperm of the Spotted scat (Scatophagus argus, Linnaeus, 1766) observed by scanning and transmission electron microscopy.

An investigation was conducted to understand the sperm cell morphology and ultrastructure of Spotted scat (Scatophagus argus) through scanning and transmission electron microscopy. The present study reveals that the sperm of S. argus can be differentiated into three major parts - an acrosome-less spherical head, a short mid-piece, and a cylindrical flagellum. The scat sperm cell had a mean total length of 21.32 ± 1.80 µm with the presence of ovoid electron-dense nucleus. The mean length and width of ovoid nucleus measured 1.44 ± 0.34 and 1.54 ± 0.33 µm, respectively. The structural characteristics of the nucleus were found to be a shallow axial nuclear fossa and centriolar complex. The two centrioles were positioned nearly perpendicular to each other with a conventional "9 + 0" pattern in the proximal centriole. The short mid-piece was located laterally to the nucleus and contains 5 or 6 spherical and unequal-sized mitochondria. The mitochondria were separated from the axoneme by a cytoplasmic canal. The flagellum was inserted at the base of the nucleus with the presence of an axoneme structure of 9 + 2 paired micro tubules. The sperm flagellum had short irregular lateral fins. The present study reveals that Spotted scat sperm can be categorized as being of a "primitive or ect-aquasperm type" and belongs to the teleostean "type I" sperm. This is the first report on the morphology and ultrastructure of sperm in Scatophagidae family.

Diversity of sulfur-oxidizing bacteria in greenwater system of coastal aquaculture.

Reduced sulfur compounds produced by the metabolism are the one of the major problems in aquaculture. In the present study, herbivorous fishes have been cultured as biomanipulators for secretions of slime, which enhanced the production of greenwater containing beneficial bacteria. The genes encoding soxB which is largely unique to sulfur-oxidizing bacteria (SOB) due to its hydrolytic function has been targeted for examining the diversity of SOB in the green water system of coastal aquaculture. Novel sequences obtained based on the sequencing of metagenomic clone libraries for soxB genes revealed the abundance of SOB in green water system. Phylogenetic tree constructed from aligned amino acid sequences demonstrated that different clusters have only 82-93% match with Roseobacter sp., Phaeobacter sp., Roseovarius sp., Sulfitobacter sp., Ruegeria sp., and Oceanibulbus sp. The level of conservation of the soxB amino acid sequences ranged from 42% to 71%. 16S rRNA gene analyses of enrichment culture from green water system revealed the presence of Pseudoxanthomonas sp., which has 97% similarity with nutritionally fastidious Indian strain of Pseudoxanthomonas mexicana-a sulfur chemolithotrophic gamma-proteobacterium. Our results illustrate the relevance of SOB in the functioning of the green water system of coastal shrimp aquaculture for oxidation of reduced sulfur compounds, which in turn maintain the sulfide concentration well within the prescribed safe levels.

Nodavirus infection causes mortalities in hatchery produced larvae of Lates calcarifer: first report from India.

Larvae (15 to 21 d post hatch, dph) of the Asian sea bass Lates calcarifer (Bloch) suffered heavy mortalities (60 to 90%) during the hatchery-rearing phase. Darkened and moribund larvae showed no evidence of bacterial or parasitic infections. Tissue sections of brain and spinal cord showed clear necrotic vacuolation. Electron microscopy revealed membrane-bound viral particles in the cytoplasm of the nerve cells. The viral particles measured 28 to 30 nm in diameter. Primer sets, designed for the amplification of the RNA2 segment of the piscine nodavirus coat protein gene, were used in the RT-PCR analysis of moribund larvae of 20 and 21 dph which produced the amplified product of 430 bp. The clinical manifestations, pathology and electron microscopy observations supported by the RT-PCR analysis suggest that the nerve necrosis was due to nodavirus infection in the larvae. This is the first report of piscine nodavirus infection from the Indian sub-continent.

Acute toxicity of some heavy metals to Lates calcarifer fry with a note on its histopathological manifestations.

Static bioassay tests for acute toxicity of four heavy metals to fry of the fish Lates calcarifer in brackishwater medium were conducted. The 96 h LC50 values of metals such as Hg, Cu, Cr, and Mn for two different size groups 11 +/- 3 mm and 24 +/- 4 mm were found to be 0.085, 1.3, 27.9, and 220 mg/L and 0.20, 1.8, 33, and 250 mg/L, respectively. The rank order of toxicity of metals was found to be Hg > Cu > Cr > Mn. Using the safe concentration factor of 0.01, the allowable safe concentration for Hg, Cu, Cr, and Mn in brackishwater are 0.00085-0.002, 0.013-0.018, 0.279-0.33, and 2.20-2.50 mg/L, respectively. The damage caused to different organ systems of the fish, exposed to the heavy metals, agrees with the rank order of the toxicity. Heavy nerve tissue necrosis was observed in fish exposed to mercury. Copper induced gill epithelial necrosis and kidney tubular degeneration. Chromium and manganese accounted for various histopathological manifestations involving vital organs such as liver and kidney.

Early alteration in glomerular reserve in humans at genetic risk of essential hypertension: mechanisms and consequences.

Essential hypertension has a familial predisposition, but the phenotype of elevated blood pressure has delayed penetrance. Because the kidney is a crucial determinant of blood pressure homeostasis, we studied early glomerular alterations in still-normotensive young subjects at genetic risk of hypertension. Thirty-nine normotensive adults (mean age 29 to 31 years), stratified by genetic risk (parental family history [FH]) of hypertension (26 with positive FH [FH+], 13 with negative FH [FH-]), underwent intravenous infusion of mixed amino acids. Before and during amino acid administration, we measured glomerular filtration rate (GFR), putative second messengers of amino acids (nitric oxide [NO.] metabolites and cGMP), serum insulin and amino acid concentrations, and the FE(Li)+ as an index of renal proximal tubular reabsorption. The FH+ group had a blunted GFR rise in response to amino acids (2.43+/-8.16% versus 31.0+/-13.4% rise, P:=0.0126). The amino acid-induced change in GFR correlated (r=0.786, P:<0.01) with the change in urinary NO. metabolite excretion; a diminished rise in urinary NO. metabolite excretion in the FH+ group (P:=0.0105) suggested a biochemical mechanism for the different GFR responses between FH groups: a relative inability to convert arginine to NO. The FH+ group had a far lower initial cGMP excretion at baseline (261+/-21.1 versus 579+/-84.9 nmol. h(-1)/1.73 m(2), P:=0.001), although cGMP did not change during the amino acid infusion (P:=0.703). FH status, baseline GFR, and baseline serum insulin jointly predicted GFR response to amino acids (P:=0.0013), accounting for approximately 45% of the variance in GFR response. Decline in FE(Li)+, an inverse index of proximal tubular reabsorption, paralleled increase in GFR (r=-0.506, P:=0.01), suggesting differences in proximal tubular reabsorption during amino acids between the FH groups. GFR response to amino acid infusion was blunted in the FH+ group despite significantly higher serum concentrations of 6 amino acids (arginine, isoleucine, leucine, methionine, phenylalanine, and valine) in the FH+ group, suggesting a novel form of insulin resistance (to the amino acid-translocating action of insulin) in FH+ subjects. We conclude that blunted glomerular filtration reserve in response to amino acids is an early-penetrance phenotype seen even in still-normotensive subjects at genetic risk of hypertension and is linked to impaired formation of NO. in the kidney. Corresponding changes in GFR and fractional excretion of Li(+) suggest that altered proximal tubular reabsorption after amino acids is an early pathophysiologic mechanism. Resistance to the amino acid-translocating actions of insulin may play a role in the biological response to amino acids in this setting. This glomerular reserve phenotype may be useful in genetic studies of renal traits preceding or predisposing to hypertension.

Plasma hydrogen peroxide production in human essential hypertension: role of heredity, gender, and ethnicity.

Oxygen free radicals, including hydrogen peroxide, may mediate oxidative stress in target organ tissues and contribute to cardiovascular complications in hypertension. To examine heritability of hydrogen peroxide production, we investigated this trait in a family-based cohort consisting of family members (n=236) ascertained through probands (n=57) with essential hypertension. Significant effects on hydrogen peroxide production were found for gender and ethnicity, with men having greater values than women (P<0.001) and white subjects having greater values than black subjects (P=0.025). Hydrogen peroxide production correlated directly with plasma renin activity (P=0.015), suggesting an important interaction between circulating oxygen radicals and the renin-angiotensin system and a potential mechanism for lower hydrogen peroxide values observed in blacks. Heritability estimates from familial correlations revealed that approximately 20% to 35% of the observed variance in hydrogen peroxide production could be attributed to genetic factors, suggesting a substantial heritable component to the overall determination of this trait. Hydrogen peroxide production negatively correlated with cardiac contractility (r=-0.214, P=0.001) and renal function (r=-0.194, P=0.003). In conclusion, these results indicate that hydrogen peroxide production is heritable and is related to target organ function in essential hypertension. Genetic loci influencing hydrogen peroxide production may represent logical candidates to investigate as susceptibility genes for cardiovascular target organ injury.

Circulating amylin in human essential hypertension: heritability and early increase in individuals at genetic risk.

BACKGROUND: Human essential hypertension is a complex trait with poorly understood genetic determination. Insulin resistance is frequently associated with this trait. OBJECTIVE: To determine whether a potentially pathogenic feature of the insulin-resistant state, circulating amylin (islet amyloid polypeptide, co-released with insulin from pancreatic islet beta-cells), is already increased in prehypertensive individuals (normotensive persons at genetic risk of hypertension because of family history), whether such individuals already differ in their amylin response to beta-cell stimulation, and whether plasma amylin concentration is heritable. Such features could establish increased circulating amylin as a hereditary 'intermediate phenotype' useful in genetic analyses of hypertension. METHODS: Plasma amylin and insulin were measured in 283 medication-free individuals stratified by blood pressure status (82 hypertensive and 201 normotensive), and genetic risk (family history) of hypertension. Differences in means were tested by ANOVA, variances by F test, and frequency distributions by maximum likelihood analysis. Co-release of amylin and insulin was provoked by intravenous infusion of mixed amino acids. The effect of antihypertensive treatment was evaluated after monotherapy with either angiotensin converting enzyme inhibition or calcium-channel blockade in hypertension. RESULTS: Plasma amylin was increased in hypertension (P= 0.027), and body mass index was a strong predictor of increased circulating amylin (P = 0.0001). Plasma amylin and plasma renin activity were not correlated (P = 0.395), and effective antihypertensive monotherapy with either angiotensin converting enzyme inhibition or calcium-channel blockade did not affect either amylin (P = 0.87-0.97) or insulin (P= 0.55-0.59). Among normotensive individuals, those at genetic risk of hypertension (with positive family history) already had increased concentrations of amylin (P< 0.001), despite exhibiting no difference in blood pressure or body mass index compared with the family-history-negative group; however, among normotensive individuals, both family history (P = 0.043) and body mass index (P= 0.0059) were significant predictors of increased concentrations of amylin. By maximum likelihood analysis, plasma amylin was distributed heterogeneously in the normotensive individuals, with two modes best explaining the distribution (chi2 = 77.4, P< 0.001), and family-history-positive individuals completely accounting for the upper mode (chi2 = 4.63, P = 0.031). Family-history-positive normotensive individuals showed greater plasma amylin concentrations both before and during beta-cell stimulation by amino acid infusion (P = 0.014). Black (n = 111) and white (n = 172) individuals did not differ in mean (P = 0.946) or variance (P = 0.172) of plasma amylin concentrations. CONCLUSIONS: These results suggest that plasma amylin concentration is in part determined by heredity. Both basal and stimulated plasma amylin excess may identify a subgroup of individuals bearing an inherited predisposition to hypertension. Measurement of amylin might identify a useful 'intermediate phenotype' in the genetic analysis of essential hypertension and its relationship to insulin resistance.

Beta2-adrenergic receptor polymorphisms at codon 16, cardiovascular phenotypes and essential hypertension in whites and African Americans.

Beta2-adrenergic receptors (beta2-AR) contribute to cardiovascular regulation by influencing several functions and previous studies suggest that a decreased function of the beta2-AR may be involved in essential hypertension. Beta2-AR are polymorphic and certain polymorphisms of these receptors are of functional importance. We focus here on the Arg16-->Gly16 beta2-AR polymorphism, which shows enhanced agonist-promoted downregulation of the receptor and which, in two recent studies, yielded opposite results in terms of association with essential hypertension: an increased frequency of the Gly16 variant in African-Caribbean hypertensives and of the Arg16 variant in offspring of Norwegian white hypertensive parents. In the current study, we genotyped 243 subjects, including both African-American and white individuals, for codon 16 polymorphism and assessed blood pressure and cardiovascular function using impedance cardiography and pressor sensitivity to phenylephrine. We found similar patterns of cardiovascular function and expression of hypertension with the two genotypes of codon 16. There was no statistically significant difference in the overall allelic distribution of the two genotypes: among African-Americans, 51% of the hypertensives and 50% of the normotensives carried the Arg16 allele, whereas among the white subjects 40% of the hypertensives and 47% of the normotensives were carriers of the Arg16 allele. Although we observed a statistically significant increase in the Arg16/Gly16 heterozygotes in the African-American population, the Gly16 allele was not significantly increased in the African-Americans compared to whites. These findings indicate that the codon 16 polymorphisms are not associated with hypertension in a mixed American study population nor do they appear to substantially impact on a variety of hemodynamic variables.

Renal kallikrein excretion: role of ethnicity, gender, environment, and genetic risk of hypertension.

BACKGROUND AND OBJECTIVE: Alterations in renal kallikrein excretion are well-described in hypertension, and kallikrein excretion may predict risk of developing hypertension, but kallikrein excretion has not been directly compared across several ethnic strata, nor have the effects of ethnicity, gender, environment, and genetic risk of hypertension been simultaneously considered as determinants of kallikrein. METHODS: We investigated determinants of kallikrein excretion in a cross-section of n = 204 normotensive subjects stratified by ethnicity (119 Caucasian, 33 African-American, 52 Asian), gender (109 men, 95 women), environment (spontaneous electrolyte intake/excretion), and heredity (genetic risk (family history) of hypertension). Results were interpreted by analysis of variance (with Bonferroni post hoc comparison corrections), analysis of covariance, multiple linear regression, and maximum likelihood. RESULTS: Urinary kallikrein activity varied substantially (F = 5.30, P = 0.006) across the three ethnic groups, with African-American values approximately 50% lower than Caucasian (P = 0.005) or Asian (P = 0.02). Ethnicity and gender (T = 3.24, P = 0.001) had independent effects on kallikrein, with women excreting approximately 50% more kallikrein than men, regardless of ethnicity. Subjects at genetic risk of hypertension were over-represented (P = 0.048) in the lower stratum of a bimodal distribution of kallikrein excretion (chi-square = 29.6, P < 0.001). Potassium excretion was diminished in African-Americans (P < 0.001 to P = 0.002), and in a multivariate analysis, potassium excretion was the strongest correlate of kallikrein excretion (T = 4.10, P = 0.0001). In a subset of Caucasian and African-American individuals, African-Americans exhibited diminished excretion of not only kallikrein and potassium, but also aldosterone (P = 0.003), suggesting a mechanistic link between potassium and kallikrein excretion in their ethnic variations. CONCLUSIONS: Kallikrein excretion is influenced by several independent determinants, both hereditary (gender, ethnicity, and genetic risk of hypertension) and environmental (potassium intake and excretion). Ethnicity and environment may interact uniquely to influence kallikrein, as demonstrated by the case of African-Americans with diminutions of both kallikrein and potassium excretion. These results suggest a mechanism whereby kallikrein excretion is diminished in African-Americans, as well as therapeutic strategies to correct this deficiency. Finally, the identified determinants of kallikrein excretion will require analytic adjustment during genetic studies of this 'intermediate phenotype' in hypertension. Journal of Human Hypertension (2000) 14, 461-468

Variability in phenylephrine response and essential hypertension: a search for human alpha(1B)-adrenergic receptor polymorphisms.

Genetic polymorphisms in drug receptors, in particular adrenergic receptors, may contribute to intersubject differences in pharmacologic response. We tested patients and first-degree normotensive and hypertensive relatives of patients with essential hypertension and found substantial intersubject variability in blood pressure response to infusion of the alpha(1)-adrenergic agonist phenylephrine. Because response to phenylephrine depends upon interaction with alpha(1B)-adrenergic receptors, we tested whether polymorphisms in this receptor contribute to the variable responses. Accordingly, we developed a polymerase chain reaction-based method, generating four exon-spanning fragments, to identify polymorphisms in the coding sequence of the two exons of the human alpha(1B)-adrenergic receptor. We sequenced the entire coding sequence of exon 1 from 51 subjects and exon 2 from 16 of these 51 subjects. Compared with the published sequence for the alpha(1B)-adrenergic receptor, we found one amino acid addition in exon 2 at position 368 (Arg) and one substitution (Arg371Gly) in all subjects. We thus suggest we have defined the correct coding sequence of the human alpha(1B) receptor. We found two "silent" polymorphisms in exon 1, one of which occurred in 3 of 51 subjects. These polymorphisms were unrelated to blood pressure status or response to phenylephrine. The 95% confidence intervals for expression of polymorphisms in exons 1 and 2 were 0 to 11%. Our data reveal that although phenylephrine response varies in humans, frequent polymorphisms in the coding sequence of the human alpha(1B)-adrenergic receptor appear not to account for this variation or for the increased blood pressure in patients with essential hypertension.

Racial differences in renal kallikrein excretion: effect of the ovulatory cycle.

BACKGROUND: Renal kallikrein excretion is diminished in essential hypertension, especially in African-Americans, and evidence exists for a major gene effect on the kallikrein phenotype. In addition, urinary kallikrein excretion differs by gender, with ovulating females having greater kallikrein excretion than males or postmenopausal females. Recent studies have shown that renal kallikrein excretion varies in females during the ovulatory cycle, with levels rising during the luteal phase and returning during the follicular phase to levels that are similar to those of males. In family studies, gender differences in urinary kallikrein excretion were present in white subjects, but not black subjects. We therefore hypothesized dysregulation of kallikrein biosynthetic responses in African-Americans. METHODS: We determined urinary kallikrein activity [chromogenic substrate S2266 (D-val-leu-arg-paranitroanilide) assay; in microU/mg creatinine] in white (N = 15) and black (N = 11) ovulating females during the ovulatory cycle. Serum progesterone, estrogen, plasma renin activity as well as urinary aldosterone, and urinary electrolytes were determined to investigate changes between mid-follicular and mid-luteal phases in the two groups. RESULTS: White and black groups were matched for age, body mass index, blood pressure, heart rate and renal function. Ovulatory cycle phases were confirmed by serum progesterone determinations, which increased significantly in whites and blacks to a comparable degree [0.84 +/- 0.14 nmol/liter (mid-follicular) to 29.77 +/- 4.70 nmol/liter (mid-luteal) in whites, 0.67 +/- 0.08 nmol/liter (mid-follicular) to 28.62 +/- 5.83 nmol/liter (mid-luteal) in blacks; P < 0.001 for cycle effect, P = NS for race effect and race X cycle interaction]. Urinary kallikrein activity increased from 623 +/- 86 microU/mg creatinine (mid-follicular) to 948 +/- 142 microU/mg creatinine (mid-luteal) in whites, but did not change in blacks during the ovulatory cycle [239 +/- 73 microU/mg creatinine (mid-follicular] to 244 +/- 41 microU/mg creatinine (mid-luteal)]. Two-way ANOVA revealed significant effects on urinary kallikrein for race (P < 0.001), cycle (P < 0.05), and race X cycle interaction (P < 0.05). Thus, white females had higher urinary kallikrein than black females, and demonstrated a significant increase in urinary kallikrein excretion during the ovulatory cycle, whereas no significant change in urinary kallikrein activity was seen in the black group. Enzyme kinetic studies and mixing studies demonstrated that these racial differences in renal kallikrein excretion were quantitative, rather than due to qualitative differences in the renal kallikrein enzyme or due to the presence of a kallikrein inhibitor. CONCLUSIONS: These results suggest pronounced blunting of menstrual cycle changes in urinary kallikrein excretion in black females. Blunted urinary kallikrein responses during the ovulatory cycle are consistent with dysregulation of renal kallikrein biosynthetic responses in African-Americans, a group at increased risk for hypertension.

Expression of altered alpha2-adrenergic phenotypic traits in normotensive humans at genetic risk of hereditary (essential) hypertension.

BACKGROUND: Essential (hereditary) hypertension is a common, though complex, trait with substantial heritability, but a still-obscure mode of inheritance. In this disorder with relatively late onset, knowledge of phenotypes with earlier penetrance would aid genetic analyses, as well as assessment of risk. OBJECTIVE: Because alpha2-adrenergic receptor alterations are among the most heritable in experimental genetic hypertension, we hypothesized enhanced expression of alpha2-adrenergic phenotypic traits in still-normotensive humans at genetic risk of hypertension. METHODS: We evaluated hemodynamic (blood pressure, cardiac output, systemic vascular resistance, stroke volume, and cardiac contractility) and biochemical (plasma drug, catecholamine, renin, and chromogranin A levels) responses to alpha2-adrenergic blockade with intravenous yohimbine in 84 normotensive subjects stratified by genetic risk of essential hypertension (67 with positive family histories and 17 with negative family histories of hypertension), as well as 18 subjects with established essential hypertension. Results were evaluated by analysis of variance, normal likelihood ratio test, and by maximum likelihood analysis for bimodality (i.e. mixtures) of response distributions. RESULTS: Blood pressure rose (P<0.001) during alpha2-adrenergic blockade, with greater response (P<0.001) in members of the hypertensive than in members of the normotensive group. Hemodynamically, the rise in blood pressure resulted from an increase in cardiac output (P<0.001), with associated increases in stroke volume (P=0.002) and cardiac contractility (P=0.006), without an overall change in systemic vascular resistance. Biochemically, plasma norepinephrine (P<0.001), epinephrine (P=0.001), and chromogranin A (P=0.02) rose, suggesting augmentation of efferent exocytotic sympathoadrenal activity. Cardiac output and stroke volume responses were correlated to increments in plasma catecholamines (especially epinephrine) for the positive group, but not for the negative group. Baseline plasma catecholamines predicted increments of stroke volume after administration of yohimbine (P=0.003-0.007) for the positive but not for the negative group. Simultaneous comparison of means and variances of cardiac output and stroke volume alpha2-adrenergic responses, by using a normal likelihood ratio test, revealed highly significant (P=0.025 to P<0.0001) differences between the groups of subjects with and without family histories of hypertension. Frequency histogram suggested that there was a bimodal distribution of responses of stroke volume to alpha2-adrenergic blockade for the normotensive group with positive family histories of hypertension; maximum likelihood analysis strongly rejected the hypothesis of a unimodal distribution, whereas the hypothesis of bimodality could not be rejected (chi2=18.4, P=0.0004). The second (exaggerated) mode of response of stroke volume to alpha2-adrenergic blockade, defined by maximum likelihood analysis, was found for 9.5% of subjects in the normotensive group with positive family histories of hypertension, and was characterized by significantly different responses of cardiac output (P=0.001), stroke volume (P<0.001), contractility (P<0.001), heart rate (P=0.03), systemic vascular resistance (P<0.001), and epinephrine (P<0.001). Even prior to alpha2-adrenergic blockade, baseline stroke volume (P=0.01), heart rate (P=0.04), systemic vascular resistance (P=0.005), and catecholamine (P=0.001-0.005) values for this subgroup were different than control values. CONCLUSIONS: We conclude that heterogeneous, bimodally distributed hemodynamic responses to alpha2-adrenergic blockade in subjects with positive family histories of hypertension suggest a discrete subgroup with early expression of perhaps Mendelian traits associated with risk of later development of hypertension. Such phenotypic traits ('intermediate phenotypes'), with earlier penetrance than hypertension itself, can be

Development and validation of a noninvasive method to determine arterial pressure and vascular compliance.

The ability not only to record automated systolic and diastolic pressure, but also to derive measurements of the rate of pressure change during the cardiac cycle, would have great potential clinical value. A new method has been developed to obtain pressure measurements at 20-ms intervals by oscillometric cuff signal pattern recognition. Derivation of noninvasive pressure measurements is based on a T tube aorta and straight tube brachial artery, and assumes that the systolic phase of the suprasystolic cuff signal and the diastolic phase of the subdiastolic cuff signal most closely approximate systolic and diastolic aortic pressures, respectively. Arterial pressures obtained by this method were compared with simultaneous invasive measurements from the thoracic aorta in 36 patients. Good agreement was observed between noninvasive and invasive methods for systolic (146 +/- 4 vs 145 +/- 5 mm Hg), diastolic (80 +/- 2 vs 77 +/- 2 mm Hg), and mean (100 +/- 3 vs 100 +/- 3 mm Hg) arterial pressures, and correlation coefficients were r = 0.94, 0.91, and 0.95, respectively. To assess the validity of measurements of the rate of pressure change, oscillometric cuff signals from a subgroup of 14 patients were analyzed in detail for the peak positive pressure derivative (dP/dt(Max)), peak negative pressure derivative (dP/dt(Min)), and time interval between peak positive and peak negative pressure derivatives [t(pp)]. Results (mean +/- SEM) were: [table in text]. The incorporation of measurements of the rate of pressure change into a physical model of the brachial artery was used to derive vascular compliance. A significant correlation was observed between vascular compliance derived from the oscillometric signal and determinations by either thermodilution or Fick methods and noninvasive pressures (n = 20, r = 0.83, p <0.001). Day-to-day variability for blood pressure and vascular compliance derived by the noninvasive method did not differ by >4%, representing a reproducible measure of vascular structure and function. We conclude that the measurement of absolute pressure and rate of pressure change show good correlation with catheter data and that vascular compliance can be reliably assessed by this new method. The technology should provide a valuable noninvasive tool for the assessment of both cardiac function and vascular properties.

Arterial compliance by cuff sphygmomanometer. Application to hypertension and early changes in subjects at genetic risk.

Abnormalities of the arterial pulse waveform reflect changes in cardiovascular structure and function. These abnormalities may occur early in the course of essential hypertension, even before the onset of blood pressure elevation. Previous studies of cardiovascular structure and function have relied on invasive intra-arterial cannulation to obtain the arterial pulse wave. We evaluated arterial structure and function using a noninvasive cuff sphygmomanometer in hypertensive (n = 15) and normotensive (n = 36) subjects stratified by genetic risk (family history) for hypertension. Using a simple physical model in which the aorta was assumed to be a T tube and the brachial artery a straight tube, we determined vascular compliance and peripheral resistance by analyzing the brachial artery pulsation signal from a cuff sphygmomanometer. Essential hypertensive subjects tended to have higher peripheral resistance (P = .06) and significantly lower vascular compliance (P = .001) than normotensive subjects. Vascular compliance correlated with simultaneously determined pulse pressure in both groups (n = 51, r = .74, P < .0001). Higher peripheral resistance (P = .07) and lower vascular compliance (P = .04) were already found in still-normotensive offspring of hypertensive parents (ie, normotensive subjects with a positive family history of hypertension) than in normotensive subjects with a negative family history of hypertension. Multivariate analysis demonstrated that both genetic risk for hypertension (P = .030) and blood pressure status (P = .041), although not age (P = .207), were significant predictors of vascular compliance (multiple R = .47, P = .011). However, by two-way ANOVA, genetic risk for hypertension was an even more significant determinant (F = 7.84, P = .007) of compliance than blood pressure status (F = 2.69, P = .089). Antihypertensive therapy with angiotensin-converting enzyme inhibitors (10 days, n = 10) improved vascular compliance (P = .02) and reduced resistance (P = .003) significantly; treatment with calcium channel antagonists (4 weeks, n = 8) tended to improve vascular compliance (P = .07) and significantly reduced peripheral resistance (P = .006). We conclude that arterial vascular compliance abnormalities detected by a noninvasive cuff sphygmomanometer reflect treatment-reversible changes in vascular structure and function. Early changes in vascular compliance in still-normotensive individuals at genetic risk for hypertension may be a heritable pathogenetic feature of this disorder.