Combined suppression of the intrarenal and circulating vasoconstrictor renin-ACE-ANG II axis and augmentation of the vasodilator ACE2-ANG 1-7-Mas axis attenuates the systemic hypertension in Ren-2 transgenic rats exposed to chronic hypoxia.

The aim of the present study was to test the hypothesis that chronic hypoxia would aggravate hypertension in Ren-2 transgenic rats (TGR), a well-defined monogenetic model of hypertension with increased activity of endogenous renin-angiotensin system (RAS). Systolic blood pressure (SBP) in conscious rats and mean arterial pressure (MAP) in anesthetized TGR and normotensive Hannover Sprague-Dawley (HanSD) rats were determined under normoxia that was either continuous or interrupted by two weeks´ hypoxia. Expression, activities and concentrations of individual components of RAS were studied in plasma and kidney of TGR and HanSD rats under normoxic conditions and after exposure to chronic hypoxia. In HanSD rats two weeks´ exposure to chronic hypoxia did not alter SBP and MAP. Surprisingly, in TGR it decreased markedly SBP and MAP; this was associated with substantial reduction in plasma and kidney renin activities and also of angiotensin II (ANG II) levels, without altering angiotensin-converting enzyme (ACE) activities. Simultaneously, in TGR the exposure to hypoxia increased kidney ACE type 2 (ACE2) activity and angiotensin 1-7 (ANG 1-7) concentrations as compared with TGR under continuous normoxia. Based on these results, we propose that suppression of the hypertensiogenic ACE-ANG II axis in the circulation and kidney tissue, combined with augmentation of the intrarenal vasodilator ACE2-ANG 1-7 axis, is the main mechanism responsible for the blood pressure-lowering effects of chronic hypoxia in TGR.

Despite similar reduction of blood pressure and renal ANG II and ET-1 levels aliskiren but not losartan normalizes albuminuria in hypertensive Ren-2 rats.

The relationship between angiotensin II (ANG II) and endothelin-1 (ET-1) is known to be complex; both peptides can initiate and potentiate the gene expression of each other. This pilot study investigated the effects of the AT(1) receptor blocker losartan or the direct renin inhibitor aliskiren on mean arterial pressure (MAP) and albuminuria and the renal ANG II and ET-1 levels. 3-month-old male Ren-2 transgenic rats (TGR) were treated either with losartan (5 mg kg(-1) day(-1)) or aliskiren (10 mg kg(-1) day(-1)) for 10 weeks. At the end of the experiment, rats were decapitated and cortical and papillary parts of kidneys were separated. Plasma and tissue ANG II levels were measured by RIA and tissue ET-1 concentrations by ELISA. In all four groups of animals ET-1 levels were lowest in renal cortex and more than 100-fold higher in the papilla. Cortical and papillary ET-1 concentrations in untreated TGR significantly exceeded those of control HanSD rats and were significantly depressed by both drugs. In both strains, papillary ANG II concentrations were moderately but significantly higher than cortical ANG II, TGR exhibited higher ANG II levels both in cortex and papilla as compared to control HanSD rats. Aliskiren and losartan at the doses used depressed similarly the levels of ANG II in cortex and papilla and reduced ET-1 significantly in the renal cortex and papilla below control levels in HanSD rats. Albuminuria, which was more than twice as high in TGR as in HanSD rats, was normalized with aliskiren and reduced by 28% with losartan, although MAP was reduced to a similar degree by both drugs. Despite similar reductions of MAP and renal ET-1 and ANG II levels aliskiren appears to be more effective than losartan, at the doses used, in reducing albuminuria in heterozygous hypertensive Ren-2 rats.

End-organ damage in hypertensive transgenic Ren-2 rats: influence of early and late endothelin receptor blockade.

The rat strain transgenic for the murine Ren-2 renin gene (TGR) is defined as a monogenic model of angiotensin II-dependent hypertension with endogenous activation of the renin-angiotensin system. Homozygous males TGR develop malignant hypertension with a strong salt-sensitive component. These animals show severe hypertension, proteinuria and high mortality. Morphological changes of renal parenchyma correspond to chronic ischemic glomerular changes. Heterozygous TGR develop only mild hypertension and thus provide a more suitable model of hypertension regarding to clinical studies. Within the renal parenchyma, secondary focal segmental glomerulosclerosis (FSGS) predominates. High-salt diet in heterozygous animals induces transition from benign to malignant phase of hypertension. In this case, ischemic glomerular changes are superimposed on preexisting secondary FSGS. In the regression model of hypertension (late-onset treatment) the effect of salt intake is attenuated. In homozygous TGR, early selective ET(A) receptor blockade decreased blood pressure and ameliorated end-organ damage. Late selective ET(A) receptor blockade reduced podocyte injury despite final severe hypertension. Survival rate was markedly improved in both regimens with ET(A) selective blockade, while there was only partial improvement with early non-selective blockade. Both bosentan and atrasentan decreased ET-1 levels in both regimens. In heterozygous TGR, early and late ET(A) treatment substantially while ET(A)/ET(B) treatment partially improved survival rate. Significant effect on BP was found with early and late ET(A) blockade, while ET(A)/ET(B) blockade had no effect. Bosentan and atrasentan similarly decreased ET-1 levels on both regimens. In conclusion, selective ET(A) receptor blockade is superior to nonselective ET(A)/ET(B) receptor blockade in attenuating hypertension and end-organ damage. Its effect is more pronounced when applied early in the life.

Albuminuria and peripheral arterial disease: results from the multi-ethnic study of atherosclerosis (MESA).

BACKGROUND: The association of albuminuria with cardiovascular disease (CVD) is increasingly recognized, but its association with peripheral arterial disease (PAD) is not well characterized in subjects with or without diabetes. METHODS: Using data from the Multi-Ethnic Study of Atherosclerosis, a cohort free of clinical vascular disease, we analyzed the cross-sectional association between albuminuria and PAD in diabetic and nondiabetic subjects. A spot urine albumin-creatinine ratio (ACR) was used to define albuminuria in two ways: presence or absence of albuminuria and the degree of albuminuria (no albuminuria defined as urine ACR<17 mg/g for men and <25mg/g for women, microalbuminuria as urine ACR 17 to 249 mg/g for men and 25 to 334 mg/g for women, and macroalbuminuria as urine ACR> or =250 mg/g for men and > or =355 mg/g for women). PAD was defined by ankle-brachial index (ABI)<0.9. RESULTS: Among the 6760 subjects, aged 45-84 years, 326 (4.8%) had prevalent PAD. Eight hundred and thirteen (12.0%) subjects had microalbuminuria and 100 (1.5%) had macroalbuminuria. Among diabetic subjects, those with albuminuria (micro- and macroalbuminuria combined) were 1.90 times more likely to have PAD (95% CI: 1.19-3.04) than those with no albuminuria. After adjusting for CVD risk factors, the odds ratio modestly attenuated to 1.65 (95% CI: 1.00-2.74). For nondiabetic subjects, there were no statistically significant associations observed in the univariable and multivariable analyses. The degree of albuminuria was not associated with PAD in either diabetic or nondiabetic subjects. CONCLUSIONS: The presence, but not magnitude of albuminuria, is an important risk factor for PAD in diabetic but not in nondiabetic subjects.

Quantification of ultraviolet protective effects of pityriacitrin in humans.

Pityriacitrin (PIT), produced by Malassezia yeasts, is an UV absorbing substance that might cause hypopigmentation in pityriasis versicolor alba. We aimed to investigate the UV protective effect of PIT in humans using in vitro and in vivo test methods. Spectrophotometry of PIT cream and the vehicle was performed in the wavelength range from 290 to 400 nm. UV transmission and the sun protection factor (SPF) were assessed for different cream formulations. Using colorimetry we evaluated erythema and pigmentation following irradiation of cream-protected and non-protected skin of healthy subjects. UVB as well as UVA transmission decreased with increasing PIT concentrations. An increase of PIT concentration of 1.25, 2.5, and 5% was associated with slightly increasing SPFs of 1.4, 1.5, and 1.7, respectively. Our in vivo tests confirmed the validity of the SPF of PIT 5% cream determined in vitro. In conclusion, the UV protective effect of PIT is all in all very weak suggesting that PIT is likely only an inferior cofactor in the development of hypopigmentation in pityriasis versicolor alba lesions following sun exposure.

Renal endothelin system and excretory function in Wistar-Kyoto and Long-Evans rats.

AIM: The role of the kidney endothelin system in the renal regulation of fluid and electrolyte excretion was investigated in Wistar-Kyoto (WKY) and Long-Evans (LE) rats in which we found previously marked differences in the renal excretory responses to endothelin A receptor blockade. METHODS: The selective endothelin A and B receptor antagonists BQ-123 (16.4 nmol kg(-1) min(-1)) and BQ-788 (25 nmol kg(-1) min(-1)) were infused i.v. for 50 min in conscious chronically instrumented WKY and LE rats and their renal function and renal endothelin system were studied. RESULTS: Without effects on glomerular filtration rate or renal blood flow, BQ-123 and BQ-788 decreased by more than 50% (P < 0.01) both urine flow rate and electrolyte excretion in WKY rats but only urine flow rate (P < 0.05) in LE rats. Endothelin-1 content, preproET-1/GPDH mRNA ratio, B(max) and K(d) of total endothelin receptors in renal cortex did not differ between the two strains. In contrast, plasma endothelin-1 concentration (0.58 +/- 0.04 vs. 1.05 +/- 0.01 femtomol mL(-1); P < 0.01), renal papillary ET-1 concentration (68 +/- 5 vs. 478 +/- 62 fmol mg(-1) protein; P < 0.01) and preproET-1/GPDH mRNA ratio (0.65 +/- 0.09 vs. 0.88 +/- 0.05; P < 0.05) as well as total endothelin receptor number in renal papilla (B(max) 5.3 +/- 0.4 vs. and 9.0 +/- 1.2 pmol mg(-1) protein; P < 0.05) were markedly lower in LE than in WKY rats. In vitro studies showed that in both strains ET(B) receptors on renal cortical membranes amounted between 65% and 67% and on papillary membranes between 85% and 88%. CONCLUSION: The present data show that the selective ET(A) or ET(B) receptor blockade differentially affects tubular water and salt handling, which becomes apparent in conditions of low renal papillary endothelin receptor number and tissue endothelin-1 concentration.

Pityriasis versicolor alba.

Pityriasis versicolor alba is a hypopigmented or depigmented variant of pityriasis versicolor characterized by maculous, partly pityriasiform, scaly depigmented lesions occurring particularly in seborrhoeic areas. Long-persisting hypopigmentation after healing of the pityriasis versicolor was first described by Gudden in 1853. Hypopigmentation and depigmentation were later differentiated as an independent variant of the disease. In 1848, Eichstedt recognized the pathogen-related character of pityriasis versicolor in its hyperpigmented form. Today it is generally accepted that the disease is caused by yeasts of the genus Malassezia, of which nine species are differentiated. It is controversial whether a single species is responsible for the disease. The pathogenesis of depigmentation has not been established. A screening effect by the scale layer as well as toxic effects on pigment synthesis by fungal metabolites have been discussed. With regard to the second mechanism, the newly discovered tryptophan-derived metabolites of M. furfur might be significant. Evidence-based data concerning the therapy of pityriasis versicolor alba do not exist. According to current recommendations, pityriasis versicolor should be rapidly treated with antimycotics, followed by ultraviolet therapy to induce maturation of existent melanosomes and accelerate repigmentation. However, depigmented lesions are difficult to improve by ultraviolet therapy.

Effects of sodium restriction and cyclooxygenase-2 inhibition on the course of hypertension, proteinuria and cardiac hypertrophy in Ren-2 transgenic rats.

The present study was performed to evaluate the effects of sodium intake and of chronic cyclooxygenase-2 (COX-2) inhibition on systolic blood pressure (SBP) in heterozygous male transgenic rats harboring the mouse Ren-2 renin gene (TGR) and in transgene-negative normotensive Hannover Sprague-Dawley (HanSD). Twenty-eight days old TGR and HanSD were randomly assigned to groups fed either normal salt (NS) or low sodium (LS) diets. COX-2 blockade was achieved with NS-398 (1 mg x kg(-1).day(-1) in drinking water). During an experimental period of 26 days, SBP was repeatedly measured by tail plethysmography in conscious animals. We found that the LS diet prevented the development of hypertension in TGR and did not change SBP in HanSD. Low sodium intake also prevented proteinuria and cardiac hypertrophy in TGR. On the other hand, irrespective of sodium intake chronic COX-2 inhibition did not alter the course of SBP in either TGR or HanSD. The present data indicate that TGR exhibit an important salt-sensitive component in the developmental phase of hypertension. They also suggest that systemic COX-2-derived prostaglandins do not act as vasodilatory counterregulatory agents in TGR in which an exaggerated vascular responsiveness to angiotensin II is assumed as the pathophysiological mechanism in the development of hypertension.

Chronic endothelin receptor blockade reduces end-organ damage independently of blood pressure effects in salt-loaded heterozygous Ren-2 transgenic rats.

The present study was performed to evaluate the role of an interaction between the endothelin (ET) and the renin-angiotensin systems (RAS) in the development and maintenance of hypertension and in hypertension-associated end-organ damage in heterozygous male and female transgenic rats harboring the mouse Ren-2 renin gene (TGR). Twenty-eight days old heterozygous TGR and age-matched transgene-negative normotensive Hannover Sprague-Dawley rats (HanSD) were randomly assigned to groups with normal-salt (NS) or high-salt (HS) intake. Nonselective ET(A)/ET(B) receptor blockade was achieved with bosentan (100 mg.kg(-1).day(-1)). All male and female HanSD as well as heterozygous TGR on NS exhibited 100 % survival rate until 180 days of age (end of experiment). HS diet in heterozygous TGR induced a transition from benign to malignant phase hypertension. The survival rates in male and in female heterozygous TGR on the HS diet were 46 % and 80 %, respectively, and were significantly improved by administration of bosentan to 76 % and 97 %, respectively. Treatment with bosentan did not influence either the course of hypertension (measured by plethysmography in conscious animals) or the final levels of blood pressure (measured by a direct method in anesthetized rats) in any of the experimental groups of HanSD or TGR. Administration of bosentan in heterozygous TGR fed the HS diet markedly reduced proteinuria, glomerulosclerosis and attenuated the development of cardiac hypertrophy compared with untreated TGR. Our data show that the ET receptor blockade markedly improves the survival rate and ameliorates end-organ damage in heterozygous TGR exposed to HS diet. These findings indicate that the interaction between the RAS and ET systems plays an important role in the development of hypertension-associated end-organ damage in TGR exposed to salt-loading.

Further characterization of pigment-producing Malassezia strains.

Reference as well as field strains of Malassezia furfur (30), M. sympodialis (49), M. globosa (52), M. obtusa (one), M. restricta (one), M. slooffiae (seven), and M. pachydermatis (373) were investigated for their ability to produce pigment and fluorochromes when tryptophan (Trp) is offered as the main nitrogen source. Only the M. furfur strains produced pigment on a pigment-inducing medium (p-medium). Remarkably, the optical activity of Trp was not significant for pigment synthesis. Other nitrogen sources that are structurally similar to Trp (gramine, tryptamine, serotonin) did not induce pigment formation. All lipophilic non-furfur species failed to grow and to form pigment on this agar. However, growth of all lipid-dependent species was achieved on a modified Dixon agar in which peptone had been substituted by an equal amount of l-Trp. Here, too, the M. furfur colonies were characterized by rapidly developing dark brown halos. Furthermore, about 11% of the M. pachydermatis strains tested produced pigment formation on the p-medium, which was enhanced by addition of d-glucose. In contrast to M. furfur, pigment formation occurred after a markedly longer incubation time (4 weeks unlike 3-5 days) with a lower yield and limited color spectrum (thin layer chromatography, TLC). The UV filter pityriacitrine recently described for M. furfur was also demonstrated for M. pachydermatis by extraction, high-performance liquid chromatography (HPLC) analysis with co-elution and mass spectroscopy. The phenotypic feature of pigment formation in some strains of M. pachydermatis may confirm recent molecular-genetic findings suggesting a relationship between some strains of M. pachydermatis and M. furfur.

Hyperosmolality induced by betaine or urea stimulates endothelin synthesis by differential activation of ERK and p38 MAP kinase in MDCK cells.

We have previously shown that cultured porcine inner medullary collecting duct cells produce endothelin (ET) which suppressed arginine vasopressin (AVP)-induced cyclic adenosine monophosphate (cAMP) generation in an autocrine/paracrine feedback-like fashion. Moreover, hyperosmolality, e.g. induced by sodium chloride and urea, stimulated ET synthesis. Since others showed that hyperosmolality also activates mitogen-activated protein (MAP) kinases and that p38 MAP kinase facilitates cellular influx of betaine to protect the cell from high extracellular solute (urea) concentrations, we were tempted to investigate a potential interaction of MAP kinases with ET production in cultured MDCK cells in response to extracellular hyperosmolality induced by betaine and urea, respectively. Increased extracellular tonicity (602 +/- 8 vs. control of 323 +/- 3 mosmol/kg H(2)O) induced by betaine stimulated ERK and, more strongly, p38 kinase activity at 0.5-2 h of incubation with a rise in ET-1 synthesis to 1,713 +/- 68 vs. 378 +/- 51 fmol/mg protein/24 h under control conditions (p < 0.01). The p38 MAP kinase inhibitor SB203580 suppressed the rise in betaine-induced ET-1 synthesis by 91% to 494 +/- 38 fmol/mg protein/24 h, whereas the MEK/ERK inhibitor U0126 suppressed it moderately by 34%. Hypertonicity induced by urea moderately stimulated ERK but not p38 MAP kinase activity at 0.5-2 h and at 24-48 h and resulted in a modest rise in ET-1 synthesis to 681 +/- 61 fmol/mg protein/24 h (p < 0.05) which was significantly suppressed by U0126 to 484 +/- 16 fmol/mg protein/24 h. These results suggest that a functional interaction between the MAP kinases ERK and p38 MAP kinase and ET-1 synthesis is involved in betaine's protection of MDCK cells in vitro which may represent an in vivo mechanism of protection from hyperosmotic stress induced by high extracellular solute concentrations.

Role of nNOS in regulation of renal function in hypertensive Ren-2 transgenic rats.

The present study was performed to evaluate the role of neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) during the developmental phase of hypertension in transgenic rats harboring the mouse Ren-2 renin gene (TGR). The first aim of the present study was to examine nNOS mRNA expression in the renal cortex and to assess the renal functional responses to intrarenal nNOS inhibition by S-methyl-L-thiocitrulline (L-SMTC) in heterozygous TGR and in age-matched transgene-negative Hannover Sprague-Dawley rats (HanSD). The second aim was to evaluate the role of the renal sympathetic nerves in mediating the renal functional responses to intrarenal nNOS inhibition. Thus, we also evaluated the effects of intrarenal L-SMTC administration in acutely denervated TGR and HanSD. Expression of nNOS mRNA in the renal cortex was significantly increased in TGR compared with HanSD. Intrarenal administration of L-SMTC decreased the glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion and increased renal vascular resistance (RVR) in HanSD. In contrast, intrarenal inhibition of nNOS by L-SMTC did not alter GFR, RPF or RVR and elicited a marked increase in sodium excretion in TGR. This effect of intrarenal L-SMTC was not observed in acutely denervated TGR. These results suggest that during the developmental phase of hypertension TGR exhibit an impaired renal vascular responsiveness to nNOS derived NO or an impaired ability to release NO by nNOS despite enhanced expression of nNOS mRNA in the renal cortex. In addition, the data indicate that nNOS-derived NO increases tubular sodium reabsorption in TGR and that the renal nerves play an important modulatory role in this process.

Renal hemodynamics in space.

Renal excretory function and hemodynamics are determined by the effective circulating plasma volume as well as by the interplay of systemic and local vasoconstrictors and vasodilators. Microgravity results in a headward shift of body fluid. Because the control conditions of astronauts were poorly defined in many studies, controversial results have been obtained regarding diuresis and natriuresis as well as renal hemodynamic changes in response to increased central blood volume, especially during the initial phase of space flight. Renal excretory function and renal hemodynamics in microgravity are affected in a complex fashion, because during the initial phase of space flight, variable mechanisms become operative to modulate the effects of increased central blood volume. They include interactions between vasodilators (dopamine, atrial natriuretic peptide, and prostaglandins) and vasoconstrictors (sympathetic nervous system and the renin-angiotensin system). The available data suggest a moderate rise in glomerular filtration rate during the first 2 days after launch without a significant increase in effective renal plasma flow. In contrast, too few data regarding the effects of space flight on renal function during the first 12 hours after launch are available and are, in addition, partly contradictory. Thus, detailed and well-controlled studies are required to shed more light on the role of the various factors besides microgravity that determine systemic and renal hemodynamics and renal excretory function during the different stages of space flight.

Revised hypothesis and future perspectives.

Results from space have been unexpected and not predictable from the results of ground-based simulations. Therefore, the concept of how weightlessness and gravity modulates the regulation of body fluids must be revised and a new simulation model developed. The main questions to ask in the future are the following: Does weightlessness induce a diuresis and natriuresis during the initial hours of space flight leading to an extracellular and intravascular fluid volume deficit? Can sodium in excess be stored in a hitherto unknown way, particularly during space flight? Why are fluid and sodium retaining systems activated by spaceflight? Why are the renal responses to saline and water stimuli in space attenuated compared with those of ground simulations? How can the effects of weightlessness on fluid and electrolyte regulation be correctly simulated on the ground? The information obtained from space may be of relevance to fluid and electrolyte balance in edematous patients.

Role of nNOS in regulation of renal function in angiotensin II-induced hypertension.

Previous studies have indicated that in normotensive rats, NO produced by neuronal NO synthase (nNOS) plays an important role in modulating tubuloglomerular feedback (TGF)-mediated afferent arteriolar constriction. It has also been shown that in angiotensin (Ang) II-infused hypertensive rats, there is a reduced ability of nNOS-derived NO to counteract this vasoconstriction. The present study was performed to (1) assess in vivo renal functional responses to intrarenal nNOS inhibition in control and Ang II-infused rats and (2) determine whether changes in renal function following nNOS inhibition are mediated by unopposed stimulation of Ang II receptor subtype 1 (AT(1)). Wistar rats were infused with either saline (SAL) or Ang II (80 ng/min) by osmotic minipumps implanted subcutaneously. Mean arterial blood pressure of SAL- and Ang II-infused rats on day 13 after implantation averaged 121+/-4 (n=28) and 151+/-5 (n=30), respectively (P<0.05). There were no differences in glomerular filtration rate (GFR) (0.68+/-0.09 versus 0.59+/-0.09 mL. min(-1). g(-1)), renal plasma flow (RPF) (2.66+/-0.31 versus 2.34+/-0.39 mL. min(-1). g(-1)), and absolute sodium excretion (0.37+/-0.07 versus 0.42+/-0.09 micromol. min(-1). g(-1)). Intrarenal infusion of SAL did not change GFR, RPF, and sodium excretion in either SAL-infused (n=7) or Ang II-infused rats (n=8). Acute intrarenal administration of the nNOS inhibitor S-methyl-L-thiocitrulline (L-SMTC; 0.3 mg/h) decreased GFR, RPF, and sodium excretion in SAL-infused rats (n=9) by 29+/-4%, 38+/-4%, and 70+/-4% compared with control values (P<0.05). The pretreatment by the AT(1) receptor antagonist candesartan (750 ng IR) in SAL-infused rats (n=7) effectively prevented the decrease in RPF (-3+/-3%) elicited by nNOS inhibition and resulted in an increase in GFR (+25+/-12, P<0.05) and a concomitant greater increase in sodium excretion (84+/-12%, P<0.05) compared with control values. In contrast, in Ang II-infused rats (n=10) intrarenal inhibition of nNOS by L-SMTC did not cause significant decreases in GFR, RPF and sodium excretion (-2+/-2%, -15+/-10%, and -14+/-10%, respectively). These results suggest that in normotensive rats nNOS-derived NO counteracts Ang II-mediated vasoconstriction in the pre- and postglomerular microcirculation. Furthermore, Ang II-infused rats exhibit an impaired ability to release NO by nNOS. Decreased nNOS activity is likely to account at least partially for the enhanced TGF responsiveness in Ang II-infused rats and thus may contribute to the maintenance of hypertension in this model.

Physiologically based pharmacokinetic model for tetrachlorobenzyltoluenes in rat: comparison of in vitro and in vivo metabolic rates.

Ugilec 141 is a technical mixture of tetrachlorobenzyltoluenes (TCBTs). It was introduced in the early 1980s as a replacement for polychlorinated biphenyls (PCBs). Based on physicochemical properties and accumulation in the environment, the use of this mixture was prohibited. To gain more insight in the toxicokinetics of these compounds in mammals, rats were exposed to a single iv bolus injection of a mixture of 3 TCBTs. At different time points after dosing, the tissue and blood concentrations of the TCBTs were determined. The adipose tissue is the main storage compartment, followed by skin and muscle. The TCBTs were rapidly eliminated from the liver and the blood, with half lives ranging from 65 to 72 h. Additionally, the tissue concentration data for all 3 TCBTs were analyzed using a physiologically based pharmacokinetic (PB-PK) model. Sensitivity analysis illustrated that the elimination of the TCBTs was not influenced by metabolism only, but also by the blood flow through the liver. Furthermore, the metabolic rates derived from the model were compared to previously reported in vitro metabolic rates. The in vitro values for the TCBTs were only a factor 2 to 3 smaller than the in vivo metabolic rates, indicating the value of in vitro techniques for a priori parameterization of PB-PK models.

Endothelin synthesis and receptors in porcine kidney.

As insufficient information on the endothelin (ET) system in the porcine kidney is available at present, we investigated renal ET-1 synthesis and ET receptors in this species. Because ET specifically affects renal and glomerular haemodynamics and distal tubular reabsorption, we studied ET-1 synthesis in isolated glomeruli and in inner medullary collecting duct (IMCD) cells and preproET-1 mRNA in renal cortex, isolated glomeruli and papillary tissue. In addition, we characterized density and properties of ET receptors in membranes from isolated glomeruli and papillary tissue. In contrast to isolated IMCD cells, which synthesized 120 +/- 11 fmol h(-1) mg-1 protein of ET-1, no such synthesis was found with isolated glomeruli in our assay system. Nevertheless, with RT-PCR preproET(-1) mRNA was clearly present in renal cortex and glomeruli as well as in papillary tissue. Glomerular membranes were found to have ET receptors with Bmax of 1.6 +/- 0.2 pmol mg-1 protein and Kd of 311 +/- 33 pmol L(-1). Using BQ-123 (10-5 M), a specific blocker of ETA receptors, we found that 58% of total receptors are ETA receptors. Thus, presumably 42% are ETB receptors (Bmax 0.7 +/- 0.1 pmol mg-1 protein; Kd 429 +/- 110 pmol L(-1)). Bosentan (10-5 M), an ETA- and ETB-receptor antagonist, blocked all ET receptors in glomerular membranes. Papillary membranes showed ET receptors with Bmax of 2.1 +/- 0.2 pmol mg-1 protein and Kd of 137 +/- 11 pmol L(-1). In the presence of BQ-123 (10-5 M) we found that all receptors are ETB receptors (Bmax 2.3 +/- 0.4 pmol mg-1 protein; Kd 162 +/- 25 pmol L(-1)). Bosentan (10-5 M) again blocked all ET receptors in papillary membranes, thus confirming our previous finding that IMCD cells possess high-affinity ETB receptors mediating the diuretic effects of ET. Thus, in the porcine kidney the ET system may act in an autocrine/paracrine manner at the glomerular as well as at the IMCD level.

Cardiac hypertrophy in the Prague-hypertensive rat is associated with enhanced JNK2 but not ERK tissue activity.

Mitogen-activated protein (MAP) kinases are important intracellular mediators for proliferation and hypertrophy and therefore may also regulate cardiomyoblast growth in hypertensive heart disease. Thus, the aim of the present study was to examine the activities of MAP kinases, namely extracellular signal-regulated kinase (ERK)1,2, c-Jun NH2-terminal kinases (JNK)1,2 and p38 MAP kinase, in myocardial tissue of 12-week-old Prague normotensive (PNR) and hypertensive rats (PHR), a model of genetic hypertension with marked cardiac hypertrophy. Systolic blood pressure was 121 +/- 5 in PNR and 208 +/- 15 mm Hg in PHR (p < 0.01). Total heart weight was 247 +/- 4 in PNR vs. 316 +/- 4 mg/100 g body weight in PHR (p < 0.01). Left and right ventricular weights were 121 +/- 5 and 53 +/- 3 in PNR vs. 168 +/- 4 (p < 0.01) and 57 +/- 2 mg/100 g body weight (n.s.) in PHR. Using anti-ERK2 Western blot analysis as well as immunocomplex ERK activity assay, we found no activation of ERK2 in left or right ventricular tissue of PHR and PNR. Similary, p38 MAP kinase phosphorylation and activity were not detectable. In contrast, Western blot analysis using antiphospho-JNK antibodies revealed in myocardial tissue of right and left ventricles significantly greater phosphorylation of JNK2 in PHR than in PNR. This finding was confirmed by immunocomplex JNK activity assay using ATF-2 as substrate, which demonstrated a significant increase in JNK activity in the left ventricle of PHR as compared to PNR (6.4 +/- 1.5 vs. 2.5 +/- 0.5 OD; each n = 5; p < 0.05). In conclusion, cardiac JNK2 seems to be regulated differently from ERK2 in this rat model. In PHR, as compared to PNR, we found enhanced activity of JNK2 in the left and right ventricles suggesting that JNK2 is involved in hypertensive cardiac disease. The rise in JNK in both ventricles may result indirectly from humoral stimuli, e.g., endothelin-1 and/or angiotensin II, and may contribute to ventricular hypertrophy in this model of spontaneous hypertension.