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The Toxicology Forum convened an international state-of-the-science workshop

Toxicokinetics and risk assessment , Toxicokinetics and risk assessment , کتابخانه دیجیتالی دانشگاه علوم پزشکی و خدمات درمانی شهید بهشتی

cAMP generated in response to β-adrenoceptor stimulation modulates excitation-contraction coupling via activation of PKA. It is now widely accepted that cAMP signalling is compartmentalised in the heart and that PDEs, the only cAMP degrading enzyme, play a central role in spatial control of signal propagation. We have previously demonstrated that PKA-I and PKA-II isoforms are selectively activated by spatially confined pools of cAMP leading to phosphorylation of specific downstream targets. It has been suggested that selective deregulation of cAMP/PKA signals in specific subcellular compartment may lead to cardiac pathology. Here we investigate cAMP signalling in an in vitro model of cardiomyocyte hypertrophy and explore the role of different PDE families in the control of the local cAMP response to β-adrenoceptor stimulation. We used a real-time FRET-based imaging approach to monitor cAMP dynamics in adult myocytes. Adenoviral vectors carrying RI or RII Epac were generated by fusing the N-terminus of the cAMP FRET reporter Epac-1 and the dimerisation-docking (DD) domain of the regulatory subunit of PKA-I or PKA-II. Adult rat cardiac myocytes expressing the cAMP reporters were treated overnight with 1μM norepinephrine (NE) as a model of in vitro cardiomyocyte hypertrophy. In control cells, upon stimulation with 100nM isoproterenol, we detected a significantly higher cAMP response in the PKA-II compartment compared to the PKA-I compartment. In NE-treated myocytes, the differences between these compartments were abolished. To investigate which PDE family may be responsible for these changes in cAMP compartmentalisation, experiments were performed in the presence of selective PDEs inhibitors. We found that when PDE2 was inhibited with BAY60-7550 in control cells the resulting increase in cAMP was significantly greater in the PKA-I than in the PKA-II compartment. In hypertrophic myocytes, PDE2 inhibition resulted in a much greater increase in cAMP in the PKA-II compartment compared to control cells. Similar results were found with selective PDE4 inhibition with rolipram. Selective inhibition of PDE3 with cilostamide showed a small but significant rise in cAMP response in the PKA-I compartment in control cells. This was not replicated in hypertrophic myocytes. Our findings indicate that spatial control of cAMP/PKA signalling is altered in this in vitro model of hypertrophy, suggesting that in hypertrophic myocytes mechanisms may be activated resulting in altered localisation and/or activity of PDEs. Further studies will be necessary to fully determine the mechanism responsible for the observed changes.

This manuscript addresses guidance in the use of kinetic and dynamic data to inform quantitatively extrapolations for interspecies differences and human variability in dose-response assessment developed in a project of the International Programme on Chemical Safety (IPCS) initiative on Harmonisation of Approaches to the Assessment of Risk from Exposure to Chemicals. The guidance has been developed and refined through a series of planning and technical meetings and larger workshops of a broad range of participants from academia, government agencies and the private sector. The guidance for adequacy of data for replacement of common defaults for interspecies differences and human variability is presented in the context of several generic categories including: determination of the active chemical species, choice of the appropriate metric (kinetic components) or endpoint (dynamic components) and nature of experimental data, the latter which includes reference to the relevance of population, route and dose and the adequacy of the number of subjects/samples. The principal objective of this guidance developed primarily as a resource for risk assessors, is to foster better understanding of the components of and criteria for adequacy of chemical-specific data to quantitate interspecies differences and human variability in kinetics and dynamics. It is anticipated that this guidance will also encourage the development of appropriate data and facilitate their incorporation in a consistent fashion in dose-response assessment for regulatory purposes (IPCS, 2001).

The U.S. Environmental Protection Agency has recognized the need to develop a framework for human health risk assessment that puts a perspective on the approaches in practice throughout the Agency. In response, the Agency's Risk Assessment Forum has begun the long-term process of developing a framework for human health risk assessment. The framework will be a communication piece that will lay out the scientific basis, principles, and policy choices underlying past and current risk assessment approaches and will provide recommendations for integrating/harmonizing risk assessment methodologies for all human health endpoints.

Boron, which is ubiquitous in the environment, causes developmental and reproductive effects in experimental animals. This observation has led to efforts to establish a Tolerable Intake value for boron. Although risk assessors agree on the use of fetal weight decreases observed in rats as an appropriate critical effect, consensus on the adequacy of toxicokinetic data as a basis for replacement of default uncertainty factors remains to be reached. A critical analysis of the existing data on boron toxicokinetics was conducted to clarify the appropriateness of replacing default uncertainty factors (10-fold for interspecies differences and 10-fold for intraspecies differences) with data-derived values. The default uncertainty factor for variability in response from animals to humans of 10-fold (default values of 4-fold for kinetics and 2.5-fold for dynamics) was recommended, since clearance of boron is 3-to 4-fold higher in rats than in humans and data on dynamic differences—in order to modify the default value—are unavailable. A data-derived adjustment of 6-fold (1.8 for kinetics and 3.1 for dynamics) rather than the default uncertainty factor of 10-fold was considered appropriate for intrahuman variability, based on variability in glomerular filtration rate during pregnancy in humans and the lack of available data on dynamic differences. Additional studies to investigate the toxicokinetics of boron in rats would be useful to provide a stronger basis for replacement of default uncertainty factors for interspecies variation.

The Safe Drinking Water Act Amendments of 1996 will bring substantial changes to the national drinking water utilities, States, and the U.S. Environmental Protection Agency (USEPA), as well as greater protection and information to the 240 million Americans served by public water systems. These changes comprise a balanced, integrated framework of reform, and represent a major national commitment to new and stronger approaches to prevent contamination; better information for consumers; and regulatory improvements, including better science, prioritization of effort, risk assessment, management and communication. Specifically, the Amendments require the use of the best available, peer‐reviewed science and supporting studies conducted in accordance with sound and objective scientific practices. The 1995 USEPA Risk Characterization Policy satisfies some of these requirements since it must feature values such as transparency in decision making process; clarity in communicating with each other and the pu...

Regulatory decisions should be made in the most expert and informed way since they are precipitated by real and perceived threats to human health, under the glare of public scrutiny. In 1994, the National Research Council (NRC) reported that the U.S. Environmental Protection Agency's (USEPA's) overall approach to assessing risks is fundamentally sound, but the Agency must more clearly establish the scientific and policy basis for risk estimates and better communicate the associated uncertainties. On March 21, 1995, USEPA issued a risk characterization policy and guidance. In this policy, an effective risk characterization must fully and clearly characterize risks and disclose the scientific analysis, uncertainties, assumptions, and science policy that underlie decisions throughout the risk assessment process. A number of regulatory reform bills which required risk characterization as part of all Federal risk assessments were introduced by the 104th Congress. The purpose of this workshop was to familiarize Society of Toxicology members with: (1) key elements to be considered in risk characterization and (2) new advances in risk characterization addressed by Federal and State agencies, industry, academia, NRC, and Presidential/Congressional Commission on Risk Assessment and Risk Management. Furthermore, the main objective was to engage the audience in discussing the proper role of science in risk assessment-risk management interface to make informed decisions in the face of scientific uncertainty.

Aluminum is a ubiquitous substance with over 4,000 uses. Aluminum, as aluminum sulfate, is commonly used in the United States as a coagulant in the treatment of drinking water. For many years aluminum was not considered to be toxic to humans. However, reports associating aluminum with several skeletal and neurological disorders in humans suggest that exposure to aluminum may pose a health hazard. In 1983 the US Environmental Protection Agency (EPA) announced plans to regulate a number of substances, including aluminum, in drinking water. Aluminum was considered because of its occurrence and apparent toxicity. Upon further evaluation of the health effects data the EPA proposed not to regulate aluminum as a result of the uncertainty of the toxicity of ingested aluminum. Putative causal associations between aluminum exposure and neurological disorders such as Alzheimer's disease have yet to be substantiated. Although several issues regarding the toxicity of ingested aluminum are unresolved, aluminum has been specified in the 1986 Amendments to the Safe Drinking Water Act, as one of 83 substances in drinking water to be regulated by 1989. Additional data are needed before the potential risk of aluminum can be assessed; therefore the EPA has deferred possible regulation until 1991.

The general approach to assessment of risk from chemical contaminants in drinking water involves three steps: hazard identification, exposure assessment, and dose-response assessment. Traditionally, the risks to humans associated with different levels of a chemical have been derived from the toxic responses observed in animals. It is becoming increasingly clear, however, that further information is needed if risks to humans are to be assessed accurately. Biologically based models help clarify the dose-response relationship and reduce uncertainty.

This article presents important issues surrounding risk assessment-for inorganic compounds in drinking water. It is not intended to be an all-inclusive document of health effects research needs for specific inorganics in drinking water, but rather a glimpse into the future toward the broad research needs in 2000 and beyond. Although many of the issues discussed are not specific to inorganics, they are addressed within this context

This review will focus primarily on ohe effects of the inorganic arsenicals (arsenate and arsenite forms) that are present in drinking water. They are acutely toxic to both humans and animals, an effect that may be related to their bioavailibility. In humans, arsenicals have been reported to cause dermatitis and mucous membrane irritation upon exposure. They have also been reported to cause skin lesions and peripheral neurotoxicity in smelter workers and in patients treated with Fowler's Solution. When humans are exposed to arsenic in drinking water, effects such as hyperkeratosis, electromyographic abnormalities and vascular effects have been reported. In experimental animals, arsenic has been demonstrated to affect the liver and kidneys. In mice, arsenic has also been reported to decrease the animal's resistance to certain viral infections. The arsenite (+3) and arsenate (+5) forms have different modes of action. Arsenite binds to sulphhydryl groups and has been reported to inhibit over 100 different enzymes, while the arsenate can substitute for phosphate in various high energy intermediates, resulting in arsenolysis. In addition, when arsenate is reduced to arsenite in the body, it can also cause toxicity as that species.

The Safe Drinking Water Act Amendments of 1986 required that the U.S. Environmental Protection Agency (EPA) establish standards for 83 contaminants by June 1989, and at least 25 added standards by January 1991, then 25 more every three years hence. Conceptually, the regulatory process employed by EPA consists of two steps. First, a detailed health risk assessment of a contaminant is performed in order to determine the Maximum Contaminant Level Goal (nonenforceable health goal) in water that should result in no known or anticipated health effects and allows adequate margin of safety. Second, the results of risk assessment and management (taking best available technology, treatment techniques, cost, and other means into consideration) are combined to derive the Maximum Contaminant Level (enforceable standard) which is set as close to the MCLG as feasible. With this overall regulatory process in mind, a detailed risk assessment process (hazard identification, dose-response assessment, human exposure assessment, and risk characterization) used in setting drinking water standards is discussed. In addition, this article discusses our efforts in exploring new and improved risk assessment methodologies addressing the mechanism of action of toxicants, relative source contribution, weight of evidence, carcinogenic potency, and toxicokinetics.

Sphingolipids and their metabolites, ceramide, sphingosine and sphingosine-1-phosphate, are involved in a variety of cellular processes including differentiation, cellular senescence, apoptosis and proliferation. Ceramide is the main second messenger, and is produced by sphingomyelinase-induced hydrolysis of sphingomyelin and by de novo synthesis. Many stimuli, e. g. growth factors, cytokines, G protein-coupled receptor agonists and stress (UV irradiation) increase cellular ceramide levels. Sphingomyelin in the plasma membrane is located primarily in the outer (extracellular) leaflet of the bilayer, whilst sphingomyelinases are found at the inner (cytosolic) face and within lysosomes/endosomes. Such cellular compartmentalisation restricts the site of ceramide production and subsequent interaction with target proteins. Glycosphingolipids and sphingomyelin together with cholesterol are major components of specialised membrane microdomains known as lipid rafts, which are involved in receptor aggregation and immune responses. Many signalling molecules, for example Src family tyrosine kinases and glycosylinositolphosphate-anchored proteins, are associated with rafts, and disruption of these domains affects cellular responses such as apoptosis. Sphingosine and sphingosine-1-phosphate derived from ceramide are also signalling molecules. In particular, sphingosine-1-phosphate is involved in proliferation, differentiation and apoptosis. Sphingosine-1-phosphate can act both extracellularly through endothelial-differentiating gene (EDG) family G protein-coupled receptors and intracellularly through direct interactions with target proteins. The importance of sphingolipid signalling in cardiovascular development has been reinforced by recent reports implicating EDG receptors in the regulation of embryonic cardiac and vascular morphogenesis.

Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKtheta, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKtheta was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKtheta translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKtheta activity in immunoprecipitates (126% at 60 s). Membrane translocation and DGKtheta activation were regulated differently: NA-induced DGKtheta activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKtheta suggests that this pathway may have a physiological role in vascular smooth-muscle responses.

It is established that variations in the structure and activities of betaI spectrin are mediated by differential mRNA splicing. The two betaI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of betaII spectrins? Thus far, only a long form of betaII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of betaII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7. 0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of betaII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of betaII spectrin isoforms correlates with their differential intracellular targeting.

Protein Kinase C (PKC) is a Ca++ and phosphatidylserine (PS) dependent serine/threonine kinase that functions as a ubiquitous cellular mediator of signal transduction initiated by a variety of agonists (1).

The general approach to assessment of risk from chemical contaminants in drinking water involves three steps: hazard identification, exposure assessment, and dose-response assessment. Traditionally, the risks to humans associated with different levels of a chemical have been derived from the toxic responses observed in animals. It is becoming increasingly clear, however, that further information is needed if risks to humans are to be assessed accurately. Biologically based models help clarify the dose-response relationship and reduce uncertainty.

The US Environmental Protection Agency prepares Health Advisories (HA) for drinking water contaminants. The HA provide technical guidance to public health officials or other interested groups on many aspects concerning drinking water contamination. The HA contain information on the chemistry, health effects, analytical methods and treatment technologies for specific contaminants. In addition, the HA include a risk assessment section which provides concentrations of the contaminant in drinking water that are not anticipated to cause adverse, noncancer health effects for 1 or 10 days or for longer exposures. Because the HA include risk assessments for less than lifetime exposures, they are useful when accidental spills occur or when regulatory limits are temporarily exceeded. The guidance documents are updated when new information becomes available that would change the previous conclusions.

Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKtheta, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKtheta was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKtheta translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKtheta activity in immunoprecipitates (126% at 60 s). Membrane translocation and DGKtheta activation were regulated differently: NA-induced DGKtheta activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKtheta suggests that this pathway may have a physiological role in vascular smooth-muscle responses.

This study was undertaken to explore the effects of chronic low-level cadmium ingestion in Dahl hypertension-resistant (R) and hypertension-sensitive (S) lines of rats. Groups of weanling female R and S rats were given 0 or 1 mg cadmium/1. in drinking water and fed either a low salt (0.4% NaCl) or a high salt (4% NaCl) diet for 28 weeks. Cadmium produced hypertension associated with gross cardiac hypertrophy and mild to moderate renal vascular changes in S, but not in R, rats on a low salt diet. Cadmium enhanced the rate and degree of development of salt-induced hypertension without exacerbating the hypercholesterolemia or renal vascular lesions normally observed in S rats on a high salt diet. Cadmium lowered circulating cholesterol levels in both lines on a low salt diet. Cadmium had no influence on growth, blood urea nitrogen concentration, plasma renin activity, tumor formation, or survivorship in R and S rats on either salt diet. This study indicates that the genetic composition is a critical determinant of the adverse effects of chronic low-level cadmium ingestion in rats. In addition to the experimental implications, these findings may have relevance to the problem of human "essential" hypertension.

Dahl hypertension-resistant (R) and hypertension-sensitive (S) rats were used to determine whether cadmium-induced hypertension is dependent on genetic predisposition. In experiment I, 16 wk-old R and S rats of both sexes were injected with two doses of cadmium (1 and 2 mg/kg body wt, ip), whereas the controls received the same volumes of saline. Hypertension and renal vascular changes were observed in cadmium-injected S rats but not in R rats. The S females appeared more sensitive than S males to the hypertensinogenic effect of cadmium. In experiment II, groups of weanling female R and S rats were given 0, 1, 2.5, 5, or 10 mg cadmium/liter drinking water and fed either a low-salt (0.4% NaCl) or a high-salt (4% NaCl) diet for 28 wk. Cadmium produced cardiac hypertrophy (1 mg cadmium/liter) and hypertension associated with renal vascular changes (1--5 mg cadmium/ liter), and it enhanced proteinuria (1–10 mg cadmium/liter) in S rats on a low-salt diet. Also, the development of salt-induced hypertension was accelerated in cadmium-fed (1 and 2.5 mg/liter) S rats. These adverse effects of cadmium were not detected in R rats on either salt diet. In experiments I and II, cadmium concentrations in the kidneys and liver of S rats were higher (P less than 0.001) than in those of R rats. These data indicate that genetic differences influence the pathogenesis of cadmium-induced hypertension.

There is an urgent need to discuss the Office of Drinking Water's standard-setting or rulemaking process since most of the researchers whose papers are presented here directly or indirectly play a crucial role in this complex undertaking. Therefore, this paper will address the research data required to support policymaking and regulatory decisions pertaining to health effects of disinfectants and disinfection by-products.

In Dahl hypertension-resistant (R) and hypertension-sensitive (S) lines of rats, an ip cadmium injection exacerbated two-kidney Goldblatt hypertension and mortality in S but not in R rats. Renal and hepatic cadmium concentrations of S rats were markedly higher than those of R rats. These observations imply that the genetic background critically affects the adverse effects of cadmium on two-kidney Goldblatt hypertension in Dahl rats.

Dahl hypertension-resistant (R) and hypertension-sensitive (S) lines of rats were used to determine whether cadmium plays an etiological role in hypertension. In Study I, weanling (3-week-old) R and S rats of both sexes were given a low-salt (0.4% NaCl) diet and were divided into two groups. Rats in the cadmium group were injected with cadmium (2 mg/kg body weight, ip), whereas the controls received identical volumes of saline. Three weeks after the first injection, no elevations of systolic blood pressure were detected. A second dose of cadmium (1 mg/kg) produced hypertension in S females but not in S males or in R rats of either sex. Also, female S cadmium rats manifested significant (p less than 0.01) mild to moderate renal vascular changes. The concentrations of cadmium in hepatic and renal tissues of S cadmium rats were significantly higher (p less than 0.001) than in R rats. In Study II, weanling (3-week-old) female S rats on a high-salt (4% NaCl) diet were given cadmium (2 mg/kg body weight, ip) at week 3 followed by second and third injections of cadmium (1 mg/kg) at weeks 6 and 23. S controls received the same volumes of saline. Cadmium enhanced the rate and the degree of salt-induced hypertension development. Pathological lesions of periarteritis nodosa in the mesenteric arteries and renal vascular lesions occurred to the same extent in the cadmium and control groups. These data indicate that differences in genetic background influence the development of cadmium-induced hypertension in weanling rats, and that cadmium exacerbates the severity of salt-induced hypertension.

A case of haemolytic anaemia with elliptocytosis is described, in which a large part of the smaller (beta) subunit of the spectrin is truncated, and has an apparent molecular weight of about 214 000 compared with about 230 000 for the normal chain. It is shown that this is not a product of adventitious proteolysis during lysis or extraction. At the same time about 35% of the total spectrin in the cells is liberated from the membrane as the dimer (which is present in normal cells to the extent of less than 10%). The truncated (beta') chain appears exclusively in this dimer fraction. The beta'-chain is incapable of phosphorylation by the endogenous cAMP-independent membrane kinase, and it may be inferred that the deleted segment of the chain contains both the spectrin self-association site and the residues normally phosphorylated. The alpha beta'-dimer is active with respect to participation in a ternary complex with its partnering proteins in the membrane cytoskeleton, F-actin and 4.1, confirming that the phosphorylation sites are not involved in the primary interaction with the other cytoskeletal proteins at the network junctions. The spectrin alpha-chain generates the terminal tryptic fragment of molecular weight 80 000 characteristic of normal spectrin, rather than the 74 000 molecular weight peptide derived from the alpha-chain in cases of hereditary elliptocytosis and pyropoikilocytosis, associated with anomalous self-association of spectrin dimer. Membrane cytoskeletons, extracted from the patient's red cells, undergo normal gelation on incubation with cAMP-independent kinase and ATP, and thus do not resemble those derived from hereditary spherocytosis cells. The properties of the anomalous spectrin resemble in most respects that described in a French family by Dhermy et al (1982).

This study was undertaken to evaluate the effect of chronic diuretic therapy with chlorothiazide on the course of salt hypertension in hypertension-resistant (R) and hypertension-sensitive (S) strains of rats. Investigation of the effects of chlorothiazide on blood pressure, 24-hour urinary 24Na and aldosterone excretion, and plasma renin activity (PRA) produced the following observations: (1) Chlorothiazide failed to prevent the development of salt hypertension in S rats. (2) After 12 weeks, S rats on high salt puls chlorothiazide exhibited a rapid fall in blood pressure to levels indistinguishable from those of S rats on low salt. (3) Chlorothiazide significantly increased urinary 24Na excretion only in S rats on high salt (P less than 0.01). (4) Chlorothiazide significantly increased PRA and urinary aldosterone excretion in both strains on low or high salt diets (P less than 0.001). (5) Morbidity and mortality of salt hypertension were alleviated by chlorothiazide treatment. The unique aspect of this study is the finding that chlorothiazide did not abolish the hypertensiogenic action of salt in S rats.