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.

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.

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.

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.

Atrial natriuretic peptide blunts the cellular effects of cyclosporine in smooth muscle.

The effect of cyclosporine A to enhance vasoconstrictor-induced calcium (Ca2+) mobilization in vascular smooth muscle cells may contribute to important side effects in cyclosporine therapy such as hypertension and nephrotoxicity. On the other hand, atrial natriuretic peptide (ANP) is known to diminish vasoconstrictor-stimulated Ca2+ mobilization. The present study, therefore, examined the interaction of cyclosporine and ANP on Ca2+ kinetics in cultured rat vascular smooth muscle cells. Intracellular free calcium concentrations ([Ca2+]i) were measured using fura-2. 45Ca2+ was used to estimate Ca2+ efflux and cellular Ca2+ influx. Preincubation of the cells with cyclosporine (10 micrograms/ml) for 12 minutes lowered basal [Ca2+]i from 48 +/- 4 to 28 +/- 3 nM (p < 0.01). However, in the presence of cyclosporine, the angiotensin II (10(-8) M)-stimulated rise of [Ca2+]i was increased from 296 +/- 22 to 460 +/- 47 nM (p < 0.001). ANP (5 x 10(-9) M) blocked the Ca2+ mobilization by angiotensin II (71 +/- 7 versus 69 +/- 7 nM, NS) and also completely inhibited the effect of angiotensin II in the presence of cyclosporine (77 +/- 5 versus 78 +/- 5 nM, NS). Basal efflux as well as angiotensin II-stimulated 45Ca2+ efflux were not altered by preincubation with cyclosporine, indicating that the effect of cyclosporine on [Ca2+]i was not due to an inhibition of 45Ca2+ efflux.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle cells.

Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dual-specificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs.

Excitation energy transfer in Rhodopseudomonas sphaeroides chromatophore membranes fused with liposomes.

The role of phospholipid in the structural organization of the light-harvesting complexes of Rhodopseudomonas sphaeroides was examined in photosynthetic (chromatophore) membrane vesicles fused with liposomes. Photochemically active preparations with progressive phospholipid enrichment up to greater than 15-fold were obtained by both polyethylene glycol- and acidic-pH-induced fusion. Their fluorescence emission at approximately 300 and 77 K was increased by 2-3.5-fold from the peripheral B800-850 antenna relative to that from the core B875 antenna. Up to 30-40% reduction in the efficiency of excitation energy transfer between B850 and B875 was also observed at 77 K suggesting a selective, phospholipid-induced dissociation of a portion of the B800-850 from the rest of the light-harvesting system.

Combined treatment of severe essential hypertension with the new angiotensin converting enzyme inhibitor ramipril.

Ramipril is a newly synthesized angiotensin converting enzyme inhibitor without a sulfhydryl group in the molecule but with a prolonged duration of action. Efficacy, tolerance and safety of this drug were evaluated in 10 patients with severe essential hypertension. After a treatment period of at least 4 weeks with the conventional antihypertensive drug combination of a diuretic and a beta-blocking agent with the vasodilator dihydralazine, their systolic and diastolic blood pressures averaged 161 +/- 6 and 111 +/- 2 mm Hg, respectively. Because diastolic blood pressure during this drug regimen was still greater than 105 mm Hg in all patients, the patients received ramipril initially at single daily doses of 5 mg in addition to their previous medication. The first dose of 5 mg ramipril resulted in a moderate but significant decrease in systolic and diastolic blood pressure in 9 of the 10 patients to 142 +/- 5 and 104 +/- 4 mm Hg (p less than 0.01), respectively, between 3 and 6 hours after drug administration. In 1 patient blood pressure was unresponsive to ramipril and 1 patient complained of nausea and vomiting within the first week of treatment with ramipril. Within the following 8-week treatment period with a once-daily intake of 5 or, if necessary, 10 mg of ramipril, diastolic blood pressure normalized in the remaining 8 patients to less than 90 mm Hg. Systolic and diastolic blood pressure averaged 130 +/- 5 and 83 +/- 2 mm Hg, respectively, at the end of the 8-week treatment period with ramipril. Severe hypotension and reflex tachycardia were not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Baroreflex setting and sensitivity in normal subjects: effects of pharmacologic inhibition of the angiotensin I converting enzyme.

Arterial blood pressure, heart rate and the response of these hemodynamic parameters to exogenous norepinephrine were investigated in healthy volunteers (daily sodium intake of 150 mmol) during a control period and after a single oral dose of 5 mg of the angiotensin I converting enzyme (ACE) inhibitor ramipril (HOE 498). Norepinephrine was infused at doses of 0.1, 0.2 and 0.3 micrograms kg-1 min-1, each for 10 minutes, during control and 3 hours after ramipril administration. Exogenous norepinephrine induced a dose-dependent increase in mean arterial blood pressure from 76.4 +/- 0.9 mm Hg during control to 85.6 +/- 1.5, 92.2 +/- 1.8 and 98.4 +/- 2.4 mm Hg, respectively. Ramipril significantly affected the baroreceptor set point with a decrease in mean blood pressure (72.1 +/- 1.7 vs 76.4 +/- 0.9 mm Hg, p less than 0.01) in the presence of unchanged heart rate (71.7 +/- 0.9 vs 73.6 +/- 1.5 min-1). Baroreceptor sensitivity, estimated by the slope of the delta blood pressure versus delta heart rate relation, was not affected by ACE inhibition. Also, the pressor effect of exogenous norepinephrine was unchanged by converting enzyme inhibition. The present results show that ACE inhibition with ramipril in sodium-replete healthy volunteers induces a decrease in blood pressure that is not accompanied by changes in heart rate, pressor sensitivity to exogenous norepinephrine or baroreceptor sensitivity.

Calcium entry and 5-HT2 receptor blockade in oliguric ischaemic acute renal failure: effects of levemopamil in conscious rats.

1. Unilateral left renal artery occlusion for 1 h in a group of 8 untreated female Sprague-Dawley rats resulted in oliguric acute renal failure (ARF) persisting for more than 6 h after reflow, i.e. after reperfusion of the kidney by removal of the arterial clamp. In a second group of 8 rats with left unilateral ARF the effects of levemopamil (L), a calcium entry blocker with 5-hydroxytryptamine2 (5-HT2) receptor antagonistic properties, were studied. Rats received L as a continuous infusion (6 mg kg-1 h-1) from 1 h before ischaemia until 6 h after reflow. 2. Endogenous creatinine clearance, an estimate of glomerular filtration rate (GFR), of left ischaemic kidneys of untreated rats was almost completely abolished and urine flow was 0.05 +/- 0.02 and 0.03 +/- 0.01 ml h-1 100 g-1 body weight (body wt.) at 2 and at 6 h of reflow, respectively. In contrast, left ischaemic kidneys of L-treated rats revealed significantly higher GFR (0.10 +/- 0.02 and 0.03 +/- 0.01 ml min-1 g-1 kidney weight (k.wt.); P < 0.01) and urine flow (0.51 +/- 0.05 and 0.15 +/- 0.04 ml h-1 100 g-1 body wt.; P < 0.05) at 2 and 6 h of reflow, respectively. 3. At 6 h of reflow, mitochondria from the cortex of left ischaemic kidneys of untreated rats showed significantly reduced ATP synthesis when compared to right intact kidneys (0.06 +/- 0.02 vs 0.26 +/- 0.02 mumol ATP mg-1 protein min-1 (P < 0.01)). In contrast, in L-treated rats, ATP synthesis of left ischaemic kidneys was largely preserved (0.17 +/- 0.01 mumol ATP mg-1 protein min-1). 4. Ischaemia of left kidneys resulted in a significant decrease in medullary Na-K-ATPase activity to 9.6 +/- 2.4 as compared to 20.4 +/- 3.7 mumol P(i) h-1 mg-1 protein in the intact right kidneys which was not prevented by L (9.4 +/- 2.4 mumol P(i) h-1 mg-1 protein). 5. In untreated rats the calcium content in cortical mitochondria from left ischaemic kidneys had risen 2 fold to 23.0 +/- 1.8 at 6 h of reflow as compared to 12.2 +/- 0.3 nmol mg-1 protein in right intact kidneys (P < 0.01). This rise in mitochondrial calcium was not significantly attenuated by treatment with L (19.9 +/- 1.7 nmol mg-1 protein). 6. The results show that L transiently converted oliguria into non-oliguria during the early phase after reflow in ischaemic ARF, i.e. after reperfusion following 1 h of complete interruption of renal perfusion. The present data suggest indirectly that the 5-HT2-antagonistic properties of L rather than its calcium channel blocking action maintains GFR at low level and protects mitochondrial function early after reflow in this model of ischaemic ARF.

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.

Fish oil fatty acids and human platelets: dose-dependent decrease in dienoic and increase in trienoic thromboxane generation.

Dietary enrichment of membrane phospholipids with n-3 (fish-oil-derived) fatty acids has attracted attention as a putative therapeutic regimen for suppression of inflammatory and coagulatory events. Use of n-3 fatty-acid-enriched lipid infusions for parenteral nutrition results in micromolar concentrations of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DCHA) in the plasma-free fatty acid fraction. We investigated the influence of free EPA and DCHA on platelet thromboxane (Tx) A2 and A3 formation by using a recently developed high performance liquid chromatography-ELISA technique for separate quantification of the stable hydrolysis products TxB2 and TxB3. Washed human thrombocytes were incubated with free arachidonic acid (AA; 1 microM), A23187 (0.1 microM) or thrombin (5 U/mL) for stimulation; all regimens provoked large quantities of TxA2 in the absence of TxA3. Simultaneous admixture of free EPA or free DCHA to the incubation medium (concentration range, 0.01-50 microM) largely suppressed platelet TxA2 generation in response to all stimuli used in a dose-dependent manner. The effective concentration with 50% influence of arachidonic acid was 4.2 microM, whereas the inhibitory concentration with 50% effect of EPA and DCHA were both in the same order of magnitude but differed with the nature of the agonist (0.2-7 microM). Platelet (co-)incubation with EPA, but not DCHA, provoked dose-dependent synthesis of n-3-lipid-derived thromboxane: kinetics of formation and absolute quantities of TxA3 approximated 20% of the respective TxA2 data upon stimulation with AA. Both EPA and DCHA dose-dependently suppressed U46619-provoked platelet aggregation. We conclude that EPA and DCHA are potent competitive inhibitors of TxA2 generation by intact platelets, with EPA acting as poor substrate and DCHA being no substrate for the cyclooxygenase/thromboxane synthase complex. Enrichment of the plasma-free fatty acid fraction with n-3 lipids may offer a therapeutic regimen to suppress the synthesis of the potent proaggregatory and vasoconstrictory agent TxA2.

Intranasal application of atrial natriuretic peptide and 1-deamino-d-arginine vasopressin in healthy volunteers. Hemodynamic, hormonal, and renal excretory effects.

In the present study we investigated the effects of an intranasal administration of 25 micrograms alpha-human atrial natriuretic peptide (alpha-hANP) dissolved in 0.2 mL 0.9% saline on renal excretory function, blood pressure (BP), heart rate (HR), and also its interaction with the renal effects of 20 micrograms deamino-d-arginine vasopressin (d-DAVP) in 10 healthy volunteers. After two 30-min control periods plasma concentrations of ANP and cGMP rose significantly from 14.8 +/- 1.8 pmol/L and 5.9 +/- 0.3 pmol/mL to 23.3 +/- 2.3 pmol/L and 6.6 +/- 0.4 pmol/mL (P less than .05), respectively within 30 min after ANP administration. The levels returned to basal values after 60 min. Urinary cGMP excretion initially rose from 17.9 +/- 3.6 to 46.8 +/- 3.7 pmol/30 min and then returned to control values, whereas plasma renin activity and plasma aldosterone concentration decreased significantly after 30 and 60 min (P less than .05). Systolic and diastolic BP declined slightly by 8% and 7%, respectively, while HR remained unaltered. Urine flow rate and sodium excretion increased by 321% and 190%, respectively (P less than .05). These changes were also significant when data were compared with a time-matched placebo study performed on the preceding day with intranasal administration of 0.2 mL 0.9% saline alone. After 48 h the protocol was repeated but 20 micrograms d-DAVP was administered intranasally 120 min before an intranasal application of 25 micrograms alpha-hANP. Within 120 min d-DAVP had reduced urine flow by approximately 50% (P less than .05), while sodium excretion remained unaltered.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitors of Na-K-ATPase in human urine: effects of ouabain-like factors and of vanadium-diascorbate on calcium mobilization in rat vascular smooth muscle cells: comparison with the effects of ouabain, angiotensin II, and arginine-vasopressin.

Endogenous ouabain-like factors (OLF) may play a role in the pathogenesis of volume-dependent hypertension by raising intracellular free calcium ([Ca2+]i) as a consequence of inhibition of the sodium pump. In previous studies we described the presence of two low molecular (Mr approximately equals 400) inhibitors of Na-K-ATPase in human urine, ie, a more polar OLF-1 and a more apolar OLF-2. We subsequently identified the active compound in OLF-2 as vanadium (V(IV))-diascorbate (Mr 416). OLF-1, OLF-2, and V-diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. In the present study we investigated the effects of urinary OLF-1, OLF-2, and V-diascorbate on calcium mobilization, ie, on [Ca2+]i in cultured rat vascular smooth muscle (VSM) cells in comparison to the effects of ouabain, angiotensin II (A II), and arginine-vasopressin (AVP). [Ca2+]i was determined by the fura-2 method. OLF-1 and OLF-2 (each approximately equals 10(-4) mol/L), obtained as single spots by thin-layer chromatography, produced a rise in [Ca2+]i in VSM cells from 45 +/- 7 to 99 +/- 22 and from 48 +/- 9 to 92 +/- 2 nmol/L (each n = 5; P < .05), respectively, after 3 min. V-diascorbate also increased [Ca2+]i slowly and dose-dependently, eg, from 56 +/- 14 to 102 +/- 15 nmol/L at a concentration of 10(-6) mol/L (n = 5; P < .05) after 3 min. A similar slow rise in [Ca2+]i from 53 +/- 10 to 185 +/- 3 nM (n = 5; P < .05) after 3 min was found with ouabain (10(-6) mol/L). As standard vasoconstrictor, All (10(-8) mol/L) rapidly increased [Ca2+]i from 23 +/- 4 to 846 +/- 50 nmol/L (n = 7; P < .01) within 30 sec. This effect was enhanced to 1,389 +/- 161 nM (n = 7; P < .01) when VSM cells were preincubated with V-diascorbate (10(-6) mol/L) for 10 min. AVP (10(-7) mol/L) also rapidly increased [Ca2+]i to 418 +/-11 nmol/L within 30 sec (n = 7; P < .01). This effect was enhanced in the presence of OLF-2 (approximately equals 10(-4) mol/L) or ouabain (10(-6) mol/L) to 523 +/- 14 and 560 +/- 19 nmol/L, respectively (each n = 7); P < .01). The calcium channel blocker verapamil, the intracellular calcium release blocker TMB-8, and the unselective cation channel blocker Ni2+ partly blunted the A II- or AVP-induced rise in [Ca2+]i and prevented the OLF-2- and V-diascorbate-induced increase in [Ca2+]i. Thus, OLF-1, OLF-2 and V-diascorbate, the active component of OLF-2, reveal effects similar to those of ouabain on [Ca2+]i in VSM cells, ie, they produce a slow rise in [Ca2+]i subsequent to inhibition of the sodium pump. The physiologic and pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.

Effects of urinary ouabain-like factors and vanadium diascorbate on calcium mobilization in porcine inner medullary collecting duct cells: comparison with the effects of ouabain and vasopressin.

It is speculated that ouabain-like factors (OLF) play a role in the pathogenesis of volume-dependent hypertension. In previous studies we isolated a more polar OLF-1 and a more apolar OLF-2 from the urine of healthy subjects after 5 days on a high sodium intake (>400 mmol/day) by gel chromatography (Sephadex G-25 and G-10) and reverse-phase HPLC. We subsequently identified the chemical structure of OLF-2 as vanadium (V(IV)) diascorbate. OLF-1, OLF-2, and vanadium diascorbate inhibited dose-dependently porcine Na-K-ATPase in vitro. Because the inner medullary collecting duct (IMCD) plays a crucial role in the long-term regulation of body fluid volume, in the present study we investigated the effects of urinary OLF-1 and OLF-2, and of vanadium diascorbate in comparison to ouabain and vasopressin (AVP) on calcium mobilization, ie, on free calcium concentration [Ca2+]i, in cultured porcine IMCD cells. [Ca2+]i was determined by the fura-2 method in IMCD cells isolated by hypotonic treatment and density gradient centrifugation from fresh porcine kidneys. Assuming an approximate molecular weight (MW) of 400 for OLF-1 and OLF-2, OLF-1 (10(-4) mol/L) produced a slow increase in [Ca2+]i from 39 +/- 10 to 169 +/- 21 nmol/L (n = 7 ) after 4 min. Similarly, OLF-2 (10(-4) mol/L) resulted in an increase in [Ca2+]i from 74 +/- 20 to 216 +/- 52 nmol/L (n = 7) after 4 min. Vanadium diascorbate (MW 403) dose-dependently increased [Ca2+]i . At a concentration of 10(-6) mol/L it increased [Ca2+]i from 46 +/- 5 to 149 +/- 9 nmol/L (n = 5) after 4 min. A similar slow increase in [Ca2+]i was found with ouabain (10(-6) mol/L), which increased [Ca2+]i from 61 +/- 22 to 180 +/- 29 nmol/L (n = 5) after 4 min in contrast to AVP (10(-7) mol/L), which rapidly increased [Ca2+]i from 48 +/- 10 to 299 +/- 32 nmol/L (n = 4) within 30 sec. Thus, OLF-1, OLF-2, and Vanadium diascorbate, the active component of OLF-2, reveal similar effects as ouabain on IMCD cells, ie, they produce a slow increase in [Ca2+]i as expected from inhibition of Na-K-ATPase. The physiologic or pathologic roles of these and additional OLF in body fluid and blood pressure regulation and in hypertension have yet to be evaluated.

Endothelin synthesis by porcine inner medullary collecting duct cells. Effects of hormonal and osmotic stimuli.

Previously, we have shown in porcine inner medullary collecting duct (IMCD) cells that endothelin (ET), probably in an autocrine fashion, suppresses arginine vasopressin (AVP)-induced synthesis of cAMP and thereby, may modify the action of AVP on IMCD fluid transport. In the present study we investigated the effects of various stimuli including extracellular tonicity on ET synthesis in porcine IMCD cells in culture. IMCD cells produced ET in a saturationlike time-dependent manner over a period of 24 h. Neither AVP (10(-7) mol/L), bradykinin (10(-7) mol/L), nor atrial natriuretic peptide (10(-7) mol/L) affected basal ET synthesis of IMCD cells at extracellular isotonicity (323 mOsm/kg H2O). The calcium ionophore A23187 (10(-7) mol/L) increased ET production by 38% within 2 h (P < .05). Preincubation for 48 h with increased osmolality in the incubation media from 323 to 600 mOsm/kg H2O by raising the concentrations of 1) NaCl (n = 6), 2) urea (n = 6), or 3) NaCl+urea (n = 6) increased ET synthesis from a control value of 225 +/- 25 pg/mg cell protein/2 h in isotonic medium to 1) 555 +/- 13 pg/mg cell protein/2 h (P < .01), 2) 354 +/- 18 pg/mg cell protein/2 h (P < .05), and 3) 448 +/- 22 pg/mg cell protein/2 h (P < .05), respectively, in hypertonic media. These data suggest that increases in papillary osmolality are associated with enhanced ET synthesis possibly involving a calcium-dependent process and attenuating AVP-dependent fluid absorption in a short-loop feedback fashion.

Endogenous natriuretic and ouabain-like factors. Their roles in body fluid volume and blood pressure regulation.

An endogenous ouabain-like sodium pump inhibitor was demonstrated originally in serum or plasma of acutely extracellular fluid volume (ECFV) expanded animals and humans. Since then numerous studies have confirmed the presence of ouabain-like factor(s) (OLF) in blood, urine, cerebrospinal fluid, and various tissues including the heart and hypothalamus. Some of these OLFs represent well-known endogenous compounds, eg, free unsaturated fatty acids, which in vitro exhibit inhibition of transepithelial sodium transport, direct inhibition of the Na-K-ATPase enzyme, displacement of 3H-ouabain from its membrane receptor, and crossreaction with a digoxin antibody. Small molecular weight (MW) OLFs of yet unknown peptidic or nonpeptidic nature, which may be of hypothalamic origin, were also detected in various animal models of hypertension and in hypertensive patients. They may play a pathophysiological role especially in salt- and volume-dependent forms of hypertension. Our results show that OLFs increase basal and vasopressin-stimulated intracellular Ca2+ release in rat vascular smooth muscle cells in culture and in human platelets similar to the newly discovered endothelin. In addition, a natriuretic factor (natriuretic hormone) was detected by bioassay in plasma and urine, whose activity changes in parallel with sodium intake. We found that this natriuretic factor is associated with small peptides with a MW of less than 1,000. It is, however, unlikely that the two biological properties, ie, the ouabain-like and natriuretic activities, reside in a single compound. A number of circulating OLFs is certainly not identical with a humoral natriuretic factor. Nevertheless, there is increasing evidence for multiple interactions between OLF and the atrial natriuretic peptide (ANP).(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of endothelin receptors and intracellular signalling in porcine inner medullary collecting duct cells.

In porcine kidneys we investigated the characteristics of endothelin (ET) receptors that are present in papillary tissue but not in glomeruli. Therefore, porcine inner medullary collecting duct (IMCD) cells were separated by Percoll density gradient centrifugation after enzymatic and hypotonic treatment of minced papillary tissue. Studies were performed in fresh cell suspensions and in cells in primary culture. Changes in cytosolic free Ca2+ concentration [Ca2+]i were measured by the use of fura-2. Optimum binding of ET-1 was obtained by incubation for 120 min at 37 degrees C, pH 7.0 when maximal protein content was 40 micrograms. Analysis with the LIGAND program showed an average number of binding sites (Bmax) of 26.0 +/- 30.5 fmol/mg protein and dissociation constant (Kd) of 90.5 +/- 28.6 pmol/L for ET-1 and Bmax of 246.9 fmol/mg protein and Kd of 162.5 pmol/L for ET-3. ET-1, 10(-9) to 10(-6) mol/L, dose dependently raised [Ca2+]i four to tenfold, respectively, from a mean basal level of 41 nmol/L. This rise was significantly attenuated by TMB-8 and by verapamil. Preincubation with Ni2+ almost completely prevented the increment in [Ca2+]i. ET-1 slightly suppressed basal and significantly attenuated arginine vasopressin (AVP)-induced cyclic adenosine monophosphate (cAMP) synthesis. Thus, porcine IMCD cells possess a single class of super high affinity ETB receptors (ETB1). ET-1 raises [Ca2+]i through release from intracellular stores, activation of L-type calcium channels and, probably to a larger extent, through stimulation of other channels, eg, T-type calcium channels or unselective cation channels.(ABSTRACT TRUNCATED AT 250 WORDS)