Effects of renal denervation on the NKCC2 cotransporter in the thick ascending limb of the loop of Henle in rats with congestive heart failure

Aim: Congestive heart failure (CHF) is associated with increased renal sympathetic nerve activity and renal sodium retention. Rats with CHF display increased expression of the Na–K–2Cl cotransporter (NKCC2) in the renal medullary thick ascending limb of the loop of Henle (mTAL), and arginine vasopressin (AVP)‐stimulated cAMP formation in mTAL segments is increased in rats with CHF. The aim of the study was to investigate the role of RSNA on cAMP formation and NKCC2 expression in mTAL in rats with CHF. Methods: Congestive heart failure was induced in male Wistar rats by ligation of the left anterior descending coronary artery. Bilateral surgical renal denervation (DNX) was performed 3 weeks later. Two weeks after DNX, mTAL segments were isolated and stimulated with AVP. Results: Congestive heart failure rats displayed increased mTAL NKCC2 expression (2.5 ± 0.5 vs. 1 ± 0.2 in Sham rats), which was abolished by DNX. Bilateral denervation decreased basal cAMP levels in unstimulated tubules from CHF rats (CHF: 12.56 ± 7.73 fmol μg^?1 protein vs. DNX‐CHF: 7.94 ± 4.33; P < 0.05), as well as from Sham rats (SHAM: 4.70 ± 1.38 vs. DNX‐SHAM: 2.36 ± 1.52; P < 0.05). mTAL segments from DNX‐CHF and DNX‐Sham rats showed decreased AVP (10^?6 m )‐stimulated cAMP formation, compared with CHF (CHF: 11.92 ± 4.89 fmol μg^?1 protein vs. DNX‐CHF: 4.68 ± 2.47; P < 0.05) and Sham (SHAM: 10.78 ± 5.59 vs. DNX‐SHAM: 4.89 ± 2.62; P < 0.05). Conclusion: These results indicate that the renal sympathetic nerves have an effect on NKCC2 expression in the mTAL and might have an effect on cAMP formation in the TAL in CHF.     

Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failure

Introduction: Vasopressin (AVP) stimulates sodium reabsorption and Na,K,2Cl‐cotransporter (NKCC2) protein level in the thick ascending limb (TAL) of Henle's loop in rats. Rats with congestive heart failure (CHF) have increased protein level of NKCC2, which can be normalized by angiotensin II receptor type‐1 (AT₁) blockade with losartan. Aim: In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT₁ receptor blockade on this system. Method: CHF was induced by ligation of the left anterior descending coronary artery (LAD). SHAM‐operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day^?1 i.p.). Results: CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10^?6 m ) stimulated cAMP levels were significantly increased in CHF rats (25.52 ± 4.49 pmol cAMP μg^?1 protein, P < 0.05) compared to Sham rats (8.13 ± 1.14 pmol cAMP μg^?1 protein), P < 0.05). Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 ± 1.93 fmol μg^?1 protein vs. Los‐CHF: 7.49 ± 1.08, P < 0.05), but not in Sham rats (SHAM: 4.66 ± 0.59 vs. Los‐SHAM: 4.75 ± 0.71). AVP‐mediated cAMP accumulation was absent in both groups treated with losartan (Los‐SHAM: 4.75 ± 0.71 and Los‐CHF: 7.49 ± 1.08). Conclusion: The results indicate that the increased NKCC2 protein level in the mTAL from CHF rats is associated with increased cAMP accumulation in this segment. Furthermore, the finding that AT₁ receptor blockade prevents AVP‐mediated cAMP accumulation in both SHAM and CHF rats suggests an interaction between angiotensin II and AVP in regulation of mTAL Na reabsorption.     

Mechanisms of Na+-K+-ATPase phosphorylation by PKC in the medullary thick ascending limb of Henle in the rat

Sodium transport correlates with varying Na⁺-K⁺-ATPase activity rates along the nephron. Whether differences in Na⁺-K⁺-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na⁺-K⁺-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na⁺-K⁺-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43±1.99 versus 2.3±0.20, respectively, P<0.01) while Na⁺-K⁺-ATPase activity was higher in mTAL (3,402±83 vs 711±158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10^–6 M decreased phosphorylation in PCT to 125±10% (P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na⁺-K⁺-ATPase. Both calcineurin inhibitor FK-506 10^–6 M and phorbol 12-myristate 13-acetate (PMA) 10^–6 M increased the degree of Na⁺-K⁺-ATPase phosphorylation (P<0.05) and inhibited Na⁺-K⁺-ATPase activity to 657±152 and 1,448±347 pmol/mm tubule per hour, respectively, in mTAL (P<0.01). Increase in [Na⁺]_i to 30, 50 and 70 mM resulted in no changes in Na⁺-K⁺-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na⁺]_i were paralleled by decreased phosphorylation (from 120±7 to 160±15% of controls, P<0.05) and increased Na⁺-K⁺-ATPase activity (from 850±139 to 1,874±203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10^–6 M decreased both Na⁺-K⁺-ATPase dephosphorylation to 41.85±9.58% (P<0.05) and Na⁺-K⁺-ATPase activity to 2,405±176 pmol/mm tubule per hour in mTAL (P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments.     

Some alterations of the leucoeyte β2-adrenoceptor/cAMP-system in patients with seasonal allergic rhinoconjunctivitis are related to disease activity

Background Disturbances of β₂-adrenoceptors are discussed as a pathogenic factor in atopic diseases. Methods In this study tbe expression and function of β₂-adrenoceptors on peripheral blood leucocytes (PBL) of seven atopic patients with seasonal rhinoconjunctivitis and their seven healthy controls was evaluated in relation to disease activity. Earlier reported data during pollen. season were now compared with data obtained from the same subjects after their allergic symptoms had subsided. Results The variables that had indicated a β₂-adrenoceptor subsensitivity in tbe patients during pollen season returned to control values, i.e. the reduced β₂-adrenoceptor affinity, the reduced β₂-adrenoceptor sensitivity, the reduced increase of intracellular cyclic adenosine monophosphate (cAMP) content upon stimulation with isoproterenol, and the reduced cAMP plasma concentration (values no longer significantly different from those of controls). However, the variable that had suggested an increase in activity of the cAMP degrading enzyme phosphodiesterase (PDE), i.e. the reduced basal intracellular cAMP content of the patients, remained reduced after the pollen season (4.9 ± 1.1 pmol/lO⁶’cells in patients vs 8.2 ^± 0.9pmol/10⁶ cells in controls: P < 0.05). There were no significant differences in β₂-adrenoceptor density between patients and controls at both investigations. Conclusions Atopic seasonal rhinoconjunctivitis is associated with various alterations in the PBL β_2-adrenoceptor/cAMP system that depend on disease activity. The reversible β₂-adrenoceptor subsensitivity is likely to be a consequence of the disease, whereas the irreversibly decreased basal intracellular cAMP content, suggested an elevated PDE activity, might be a basic trait of atopy.     

Effects of antidiuretic hormone,parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP, 10⁻¹⁰mol.l⁻¹), parathyroid hormone (PTH, 10⁻⁸ mol.l⁻⁸) and glucagon (10⁻⁸ mol.l⁻¹) on the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were tested in in vitro perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. When compared with mTAL segments (PD_te: 8.5±0.4 mV,n=16), cTAL segments displayed a high PD_te of 15.7±0.9 mV (n=11) at the beginning of perfusion experiments which reached a value of 9.4±0.6 mV (n =11) after 38±4 min perfusion. Simultaneously R_te increased significantly from 24±3 to 28±1 Ω cm² (n=11). When PTH, AVP or glucagon were added to the bath solution, PD_te increased with PTH from 10.3±0.8 to 15.2±0.8 mV (n=13), with AVP from 10.2±0.5 to 15.0±0.7 mV (n=24) and with glucagon from 11.3±1.9 to 15.3±2.1 mV (n=8). At the same time R_te decreased from 30±3 to 23±2 Ω cm², from 28±1 to 23±1 Ω cm² and from 23±2 to 18±2 Ω cm², respectively. In mTAL segments, AVP and glucagon increased PD_te from 8.4+0.5 to 13.5±0.9 mV (n=11) and from 8.8±0.6 to 12.8±0.6 mV (n=8) respectively, while R_te decreased significantly from 23±1 to 20±1 Ω cm² and from 27±3 to 21±3 Ω cm². PTH, on the other hand, had no effect on PD_te and R_te. Since the response to PTH appeared to be specific to cTAL segments, paired experiments were performed, in which AVP or glucagon were successively tested with PTH on cTAL and mTAL segments, to ascertain the specificity of the hormonal response. In cTAL segments, PTH and AVP increased the equivalent short-circuit current (I_sc=PD_te/R_te) by 82% and 86% respectively, while PTH and glucagon, in another series, increased I_sc by 95% and 81% respectively. In mTAL segments, I_sc was increased in the presence of AVP and glucagon by 88%, and 93% respectively, whereas PTH had no effect. These results indicate that Nacl reabsorption in cTAL segments is stimulated by AVP, PTH and glucagon and in mTAL segments by AVP and glucagon. The amplitude of the response to the hormones is similar in the two segments. The residual stimulation in cTAL segments, however, persists longer than in mTAL segments.     

Quantitative MRI in murine radiation‐induced rectocolitis: comparison with histopathological inflammation score

Murine radiation‐induced rectocolitis is considered to be a relevant animal model of gastrointestinal inflammation. The purpose of our study was to compare quantitative MRI and histopathological features in this gastrointestinal inflammation model. Radiation rectocolitis was induced by localized single‐dose radiation (27 Gy) in Sprague‐Dawley rats. T₂‐weighted, T₁‐weighted and diffusion‐weighted MRI was performed at 7 T in 16 rats between 2 and 4 weeks after irradiation and in 10 control rats. Rats were sacrificed and the histopathological inflammation score of the colorectal samples was assessed. The irradiated rats showed significant increase in colorectal wall thickness (2.1 ± 0.3 mm versus 0.8 ± 0.3 mm in control rats, P < 0.0001), normalized T₂ signal intensity (4 ± 0.8 versus 2 ± 0.4 AU, P < 0.0001), normalized T₁ signal intensity (1.4 ± 0.1 versus 1.1 ± 0.2 AU, P = 0.0009) and apparent and pure diffusion coefficients (ADC and D) (2.06 × 10^?3 ± 0.34 versus 1.51 × 10^?3 ± 0.23 mm²/s, P = 0.0004, and 1.97 × 10^?3 ± 0.43 mm²/s versus 1.48 × 10^?3 ± 0.29 mm²/s, P = 0.008, respectively). Colorectal wall thickness (r = 0.84, P < 0.0001), normalized T₂ signal intensity (r = 0.85, P < 0.0001) and ADC (r = 0.80, P < 0.0001) were strongly correlated with the histopathological inflammation score, whereas normalized T₁ signal intensity and D were moderately correlated (r = 0.64, P = 0.0006, and r = 0.65, P = 0.0003, respectively). High‐field MRI features of single‐dose radiation‐induced rectocolitis in rats differ significantly from those of control rats. Quantitative MRI characteristics, especially wall thickness, normalized T₂ signal intensity, ADC and D, are potential markers of the histopathological inflammation score.     

Effect of prostaglandin E2 on agonist-stimulated cAMP accumulation in the distal convoluted tubule isolated from the rabbit kidney

The effects of calcitonin, vasoactive intestinal peptide (VIP), parathyroid hormone (PTH) and isoprenaline on intracellular cAMP accumulation were determined in the distal tubule (DCT) microdissected from collagenase-treated rabbit kidney. In DCTb (the initial bright portion) calcitonin (10 ng/ml) elicited a highly reproducible response 203.7±19.1 fmol cAMP mm^–1 4 min^–1 (SE, N=13) whereas VIP-induced cAMP accumulation was less and more variable from one experiment to another (1 M, 97.2±17.8 fmol mm^–1 4 min^–1, SE, N=12). When used in combination, these two agonists were non-additive, indicating stimulation of a single pool of cAMP in DCTb. In DCTg, (granular) which consists of at least two cell types, PTH (100 nM) elicited a marked, reproducible accumulation of cAMP (154.3±27.0 fmol mm^–1 4 min^–1; SE, N=5). Isoprenaline (1 M) and VIP (1 M) induced much smaller increases in cAMP levels 20.9±2.7 and 29.4±4.1 fmol mm^–1 4 min^–1 (SE, N=5) respectively, and, when used in combination, were non-additive, demonstrating that VIP and isoprenaline are active on the same cell type. In DCTb, prostaglandin E₂ (PGE₂) inhibited both calcitoninand VIP-stimulated cAMP accumulation (calcitonin 57.8±2.7% inhibition, SE, N=16; VIP, 80.6±2.1% inhibition, SE, N=5). The EC₅₀ values for calcitonin were 1.21±0.33 ng/ml and 1.83±0.25 ng/ ml (SD, N=3) in the absence and presence of PGE₂ (300 nM) respectively with an IC₅₀ for PGE₂ of 26.3±6.3 nM (SE, N=4). In contrast, no effects of PGE₂ were seen in DCTg vis à vis PTH, isoprenaline or VIP. The percentage inhibition of calcitonin-stimulated cAMP accumulation by PGE₂ was of the same order in the presence of isobutylmethylxanthine (an inhibitor of all types of phosphodiesterase), Ro 20-1724 (inhibitor of low-K _m cAMP-specific phosphodiesterase) or in the absence of inhibitor. Preincubation of DCTb with pertussis toxin for up to 8 h in different experimental conditions did not relieve the inhibition by PGE₂. Protein kinase C activation by phorbol ester did not attenuate calcitonin responses. These data demonstrate that the inhibition by PGE₂ of cAMP production is restricted to the initial portion (DCTb) of the distal convoluted tubule and is effective on both calcitonin and VIP responses. When tested in the presence of Ro 20-1724, ionomycin, A₁-adenosine, ₂-adrenergic and muscarinic agonists were without effect on calcitoninand PTH-stimulated cAMP accumulation in DCTb and DCTg respectively.     

Diffusion tensor imaging of renal ischemia reperfusion injury in an experimental model

Renal ischemia reperfusion injury (IRI) is a major cause of acute renal failure. It occurs in various clinical settings such as renal transplantation, shock and vascular surgery. Serum creatinine level has been used as an index for estimating the degree of renal functional loss in renal IRI. However, it only evaluates the global renal function. In this study, diffusion tensor imaging (DTI) was used to characterize renal IRI in an experimental rat model. Spin‐echo echo‐planar DTI with b‐value of 300 s/mm² and 6 diffusion gradient directions was performed at 7 T in 8 Sprague‐Dawley (SD) with 60‐min unilateral renal IRI and 8 normal SD rats. Apparent diffusion coefficient (ADC), directional diffusivities and fractional anisotropy (FA) were measured at the acute stage of IRI. The IR‐injured animals were also examined by diffusion‐weighted imaging with 7 b‐values up to 1000 s/mm² to estimate true diffusion coefficient (D_true) and perfusion fraction (P_fraction) using a bi‐compartmental model. ADC of injured renal cortex (1.69 ± 0.24 × 10^?3 mm²/s) was significantly lower (p < 0.01) than that of contralateral intact cortex (2.03 ± 0.35 × 10^?3 mm²/s). Meanwhile, both ADC and FA of IR‐injured medulla (1.37 ± 0.27 × 10^?3 mm²/s and 0.28 ± 0.04, respectively) were significantly less (p < 0.01) than those of contralateral intact medulla (2.01 ± 0.38 × 10^?3 mm²/s and 0.36 ± 0.04, respectively). The bi‐compartmental model analysis revealed the decrease in D_true and P_fraction in the IR‐injured kidneys. Kidney histology showed widespread cell swelling and erythrocyte congestion in both cortex and medulla, and cell necrosis/apoptosis and cast formation in medulla. These experimental findings demonstrated that DTI can probe both structural and functional information of kidneys following renal IRI. Copyright © 2010 John Wiley & Sons, Ltd.     

Altered basal and adenosine-mediated protein flux from coronary arterioles isolated from exercise-trained pigs

Solute flux per unit surface area and concentration gradient, (J_S/SΔC), was quantified in arterioles isolated from hearts of sedentary (SED) and exercise-trained (EX) female Yucatan Miniature Swine. Apparent permeability (P_S) was assessed from measures of J_S/SΔC for two proteins, α-lactalbumin (α-lact) and porcine serum albumin (PSA), under basal conditions and following 5 min suffusion with 10^?5M adenosine (ADO). Both proteins were labelled with the fluorescent dye tetramethyl rhodamine isothiocyanate. Basal P_s to α-lact differed with exercise training ((P^α-lact_s)_SED=5.2±1.8 (median±median absolute deviation (MAD), n=9 pigs) versus (P^α-lact_s)_EX=7.4±1.1×10^?7 cm s^?1, n=9, P<0.05). For the larger protein PSA, basal P_s did not change with training ((P^PSA_s)_SED=5.0±1.6, N=11 vs. (P^PSA_s)_EX=4.1±1.2×10^?7 cm s^?1, N=11). Suffusion of the arterioles (33±4 μm diameter, n=18 vessels) from SED hearts (n=14) with 10^?5M ADO decreased P^α-lact_s 15±8% relative to control and was without effect on P^PSA_s. By contrast, in arterioles (39±4 μm diameter, n=22 vessels) from EX hearts (n=14), ADO increased P^α-lact_s and P^PSA_s by 32 and 65% respectively, indicating that receptor-mediated changes in permeability were also sensitive to exercise training. These data demonstrate that, for coronary arterioles, permeability to macromolecules adapts to exercise training. The adaptive mechanisms may involve more than one structural component of the vessel wall as the changes in permeability were size-dependent.     

Regulation of 5-hydroxytryptamine-induced calcium mobilization by cAMP-elevating agents in cultured canine tracheal smooth muscle cells

The effects of increases in cellular adenosine 3′5′-cyclic monophosphate (cAMP) on 5-hydroxytryptamine-(5-HT-) induced generation of inositol phosphates (IPs) and increases in intracellular Ca²⁺ ([Ca²⁺]_i) were investigated using canine cultured tracheal smooth muscle cells (TSMCs). Cholera toxin and forskolin induced concentration- and time-dependent cAMP formation with half-maximal effects (−logEC₅₀) produced at concentrations of 7.0 ± 0.5 and 4.9 ± 0.4  respectively. Pretreatment of TSMCs with either forskolin or dibutyryl cAMP inhibited 5-HT-stimulated responses. Even after treatment for 24h, these agents still inhibited the 5-HT-induced Ca²⁺ mobilization. The inhibitory effects of these agents produced both depression of the maximal response and a shift to the right of the concentration response curves of 5-HT. The water-soluble forskolin analogue L-858051 [7-deacetyl-7β-(γ-N-methylpiperazino)-butyryl forskolin] significantly inhibited the 5-HT-stimulated accumulation of IPs. In contrast, the addition of 1,9-dideoxy forskolin, an inactive forskolin analogue, had little effect on this response. Moreover, SQ-22536 [9-(tetrahydro-2-furanyl)-9-H-purin-6-amine], an inhibitor of adenylate cyclase, and both H-89 [N-(2-aminoethyl)-5-isoquinolinesulphonamide] and HA-1004[N-(2-guanidinoethyl)-5-isoquinolinesulphonamide], inhibitors of cAMP-dependent protein kinase (PKA), attenuated the ability of forskolin to inhibit the 5-HT-stimulated accumulation of IPs. These results suggest that activation of cAMP/PKA was involved in these inhibitory effects of forskolin. The AlF₄ ⁻-induced accumulation of IPs was inhibited by forskolin, suggesting that G protein(s) are directly activated by AlF₄ ⁻- and uncoupled from phospholipase C by forskolin treatment. These results suggest that activation of cAMP/PKA might inhibit the 5-HT-stimulated phosphoinositide breakdown and consequently reduce the [Ca²⁺]_i increase or inhibit both responses independently. Received: 14 March 1996/Accepted: 10 April 1996     

Evidence for interference of vitamin D with PTH/PTHrP receptor expression in opossum kidney cells

 Vitamin D counters the phosphaturic action of parathyroid hormone (PTH) in rats in vivo. The present study was undertaken to examine this interaction using monolayers of Opossum kidney (OK) cells. ³²P uptake, cAMP generation, PTH/PTHrP receptor mRNA expression and intracellular Ca²⁺ [Ca²⁺]_i were measured in (1) control cells, (2) cells exposed to PTH, (3) cells pretreated with 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], and (4) 1,25(OH)₂D₃-pretreated cells exposed to PTH. ³²P uptakes were in (1) 5.00±0.20 (mean ±SE), in (2) 2.30±0.14 (P<0.001 versus group 1), in (3) 4.80±0.24 (P NS versus group 1) and in (4) 3.70±0.20 (P<0.001 versus group 2) nmol P_i/(mg·prot 10 mm). cAMP levels were in (1) 10±3, in (2) 210±8, in (3) 12±4, and in (4) 122±12 pmol cAMP/mg protein (P<0.001 versus group 2). PTH/PTHrP receptor mRNA expression was in relative units: (1) 100±0, (2) 99.5±6.2, (3) 68.7±2.6 (P<0.001 versus group 1), and (4) 34.8±3.3 (P<0.001 versus group 1). In groups 2 and 4 PTH induced equal transient increments in [Ca²⁺]_i. These experiments demonstrate that the effect of vitamin D on phosphate transport is associated with a commensurate diminution in PTH/PTHrP receptor gene expression and PTH-induced cAMP formation but not with Ca²⁺ transients. Vitamin D per se does not affect ³²P uptake or cAMP generation while it slightly decreased PTH/PTHrP receptor gene expression. These observations demonstrate that: (1) 1.25(OH)₂D₃ directly antagonizes the effects of PTH on ³²P uptake in OK cells, (2) this effect is mediated via inhibition of PTH-induced activation of AC/cAMP system, (3) the diminution in PTH-induced cAMP formation may stem at least in part from a decrease in the expression of PTH/PTHrP receptor mRNA. Received: 2 December 1997 / Received after revision: 19 January 1998 / Accepted: 28 January 1998     

Contribution of pH,diprotonated phosphate and potassium for the reflex increase in blood pressure during handgrip

The relative importance of pH, diprotonated phosphate (H₂PO₄^?) and potassium (K⁺) for the reflex increase in mean arterial pressure (MAP) during exercise was evaluated in seven subjects during rhythmic handgrip at 15 and 30% maximal voluntary contraction (MVC), followed by post-exercise muscle ischaemia (PEMI). During 15% MVC, MAP rose from 92 ± 1 to 103 ± 2 mmHg, [K⁺] from 4.1 ± 0.1 to 5.1 ± 0.1 mmol L^?1, while the intracellular (7.00 ± 0.01 to 6.80 ± 0.06) and venous pH fell (7.39 ± 0.01 to 7.30 ± 0.01) (P < 0.05). The intracellular [H₂PO₄^?] increased 8.4 ± 2 mmol kg^?1 and the venous [H₂PO₄^?] from 0.14 ± 0.01 to 0.16 ± 0.01 mmol L^?1 (P < 0.05). During PEMI, MAP remained elevated along with the intracellular [H₂PO₄^?] as well as a low intracellular and venous pH. However, venous [K⁺] and [H₂PO₄^?] returned to the level at rest. During 30% MVC handgrip, MAP rose to 130 ± 3 mmHg, [K⁺] to 5.8 ± 0.2 mmol L^?1, the intracellular and extracellular [H₂PO₄^?] by 20 ± 5 mmol kg^?1 and to 0.20 ± 0.02 mmol L^?1, respectively, while the intracellular (6.33 ± 0.06) and venous pH fell (7.23 ± 0.02) (P < 0.05). During post-exercise muscle ischaemia all variables remained close to the exercise levels. Analysis of each variable as a predictor of blood pressure indicated that only the intracellular pH and diprotonated phosphate were linked to the reflex elevation of blood pressure during handgrip.     

cAMP increases the basolateral Cl−-conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse

The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10^–9+dbcAMP 4·10^–4+forskolin 10^–6 mol·l^–1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7±0.3 mV to +12.0±0.4 mV (n=47); b) a decrease in transepithelial resistance from 25±1 cm² to 20±1 cm² (n=47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n=36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27±0.005 to 0.15±0.06 (n=12). Furosemide (10^–4 mol·l^–1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarizedPD _bl to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na⁺2Cl^–K⁺ contransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance.Supported by Deutsche Forschungsgemeinschaft Gr 480/6-2Parts of this study have been presented at the 59th Meeting of the German Physiological Society in Dortmund 1984 and at the 69th FASEB Meeting in Anaheim 1985     

Evidence that prejunctional adenosine receptors regulating acetylcholine release from rat hippocampal slices are linked to an N-ethylmaleimide-sensitive G-protein,but not to adenylate cyclase or dihydropyridine-sensitive Ca2+-channeIs

Electrically evoked [³H]acetylcholine ([³H]ACh) release from slices of the rat hippocampus was reduced in a dose-dependent manner by the adenosine A,-receptor agonist R-phenylisopropyladenosine (R-PIA) in the concentration range 0.1–10 μM. The maximal effect was observed with I μM R-PIA. Treatment with N-ethylmaleimide (NEM, 100 μ M, 10 min), which inactivates nucleotide-binding proteins (G-proteins), caused a slight increase in the basal overflow (0.17 ± 0.01%v. 0.10 ± 0.003% in the control slices), but did not affect stimulated release (0.73 ± 0.05%vs. 0.74 ± 0.03% in the control slices). N-ethylmaleimide pretreatment significantly reduced the prejunctional inhibitory effect of R-PIA on [³H]ACh release in a non-competitive manner. The S₂/S₁ ratio was 0.92 ± 0.03 in controls and was reduced to 0.32 ± 0.02 by I μ Mm R-PIA in the control slices and to 0.57 ± 0.03 after NEM pretreatment. Stimulation of cyclic AMP-accumulation by forskolin (I μ M) and rolipram (30 μ M) before the second stimulation (S₂) enhanced the S₂/S₁ ratio by about 30% to 1.26 ± 0.12, but did not reduce the inhibitory effect of R-PIA (I μ MM). The Ca²⁺-channel agonist Bay K 8644 (I μ MM), a concentration that increases K⁺-evoked noradrenaline release, did not affect the basal or electrically evoked [³H]ACh overflow, or the prejunctional effects of R-PIA (0.1 and I μ MM) on [³H]ACh release. Our results suggest that the presynaptic inhibitory effects of A₁-receptor agonists on [³H]ACh release are exerted via a nucleotide-binding protein that can be inhibited by NEM. However, the inhibitory effect is apparently not caused by a change in adenylate cyclase activity or by affecting dihydropyridine-sensitive Ca²⁺-channels.     

[Arginine]vasopressin hydrolyses phosphoinositides in the medullary thick ascending limb of mouse nephron

NaCl reabsorption across the thick ascending limb of Henle's loop (TAL) is stimulated by several hormones, in particular vasopressin acting through V₂ receptors and cyclic AMP production. This study used suspensions of medullary TAL (mTAL) tubules from the mouse nephron to investigate the possibility that, besides activating adenylyl cyclase, vasopressin also stimulates phospholipase C via V₁ receptor occupancy. Two different methods, phosphoinositide labelling and inositol trisphosphate (InsP ₃) radioimmunoassay, were used to show that [arginine]vasopressin (AVP) rapidly stimulated the formation of InsP ₃, which peaked at 200%–250% of control within the first minute of incubation with 10 nmol/l vasopressin at 37°C, and declined to basal level after 5–10 min. Dose/response curves for InsP ₃, established at 30°C and 37° C using radioimmunoassay, showed a half-maximal stimulation of InsP ₃ production at about 1 nmol/l AVP and a maximal response at 10 nmol/l. Similar values were obtained for the response to AVP in terms of cAMP accumulation. InsP ₃ content in the presence of higher concentrations of AVP (1 mol/l) was significantly lower (P<0.001) than in the presence of 10 nmol/l AVP, giving a bell-shaped appearance to the dose/response curve at 37° C but not at 30° C. The V₂ receptor agonist, 1-deamino-[8DArg]vasopressin (dAVP) did not stimulate the formation of InsP ₃, and the V₁ receptor antagonist d(CH₂)₅[Tyr(Me)²]AVP inhibited AVP-induced InsP ₃ formation, which therefore appeared to be mediated by V₁ receptor occupancy. Under the same conditions, AVP also induced the formation of diradylglycerol via V₁ receptor activation, with an analogous dose/response curve. These results show that AVP, in addition to its well-known action through V₂ receptors, also acts on the mouse mTAL through a V₁-mediated stimulation of phospholipase C. Cyclic AMP controls this transduction pathway: dAVP (10 nmol/l), dibutyryl-cAMP (1 mmol/l and 0.1 mmol/l) and forskolin (1 mol/l) decreased the InsP ₃ formation induced by AVP. Dibutyryl-cAMP itself at 37°C also reduced the diglyceride content.     

Serum eosinophil peroxidase (EPO) levels in asthmatic patients

Eosinophil granular proteins are a useful eosinophilic activation marker in asthmatic patients. In this study, the eosinophil peroxidase (EPO) levels were assessed in different stages of bronchial asthma, in 123 patients suffering from asthma, classified as mild (n=49), moderate (n=49), and severe (n=25), according to the International Consensus Report on Diagnosis and Treatment of Asthma, as well as in 27 healthy controls, with the aim of evaluating the importance of this protein as a severity marker in bronchial asthma, and its possible correlation with parameters such as anamnesis, respiratory function tests, and peripheral blood eosinophil count, and also with some allergologic diagnostic tests, both in vivo and in vitro. The geometric mean serum level of EPO was 9.3±11.3 ng/ml (median±SD) in controls, and 28±37.8 ng/ml in the asthmatic patients. Depending on the asthma severity, the EPO levels were 25±30.5; 29±37.1, and 41 ±47.3 ng/ml in mild, moderate, and severe asthmatics, respectively, being the significant differences between the group of patients with mild and severe asthma (P<0.001). The number of eosinophils (eos) in peripheral blood was 157±20 eos/mm³ in the controls, 334+35 eos/mm³ in mild asthmatics, 510 ±87 eos/mm³ in moderate asthmatics, and 658±72 eos/mm³ in severe asthmatics, with significant differences between all the groups (from P<0.05 to P<0.001). Both the serum levels of EPO and the number of eosinophils were greater in patients with active asthma than in patients with inactive asthma (P<0.001). Significant negative correlations (P < 0.001) were found between serum levels of EPO and FEY, (r_s= 0.30), MEF_25-75 (r_s= -0.33), and MEF₅₀ (r_s= -0.34), and a good positive correlation (r_s= 0.80, P<0.001) was found between EPO levels and the number of eosinophils in peripheral blood. We also found a significant positive correlation between eosinophil number and clinical score (r_s= 0.54, P<0.001) and between EPO levels and the mentioned score (r_s= 0.46, P<0.001).     

Calcium and calcimimetics regulate paracellular Na+ transport in the thin ascending limb of Henle’s loop in mouse kidney

The effect of Ca²⁺ and calcimimetics on NaCl transport was investigated in the in vitro isolated microperfused mouse thin ascending limb of Henle’s loop. In the presence of a transmural NaCl gradient, the transepithelial diffusional potential was 13.7?±?0.4 mV (n?=?17). When the Ca²⁺ in the bath was increased from 1.5 to 4.5 mM at 37°C, the relative permeability of Na⁺ to Cl^? (P _Na /P _Cl) estimated from the diffusional voltage deflection due to the transepithelial NaCl gradient (V _d) changed from 0.371?±?0.017 to 0.341?±?0.015 (n?=?10, P?<?0.0001). When the Ca²⁺ in the lumen was increased from 1.5 to 4.5 mM, the P _Na /P _Cl decreased from 0.349?±?0.013 to 0.330?±?0.013 (n?=?5, P?<?0.002). The addition of 0.1 mM neomycin and 0.2 mM gentamicin to the bath or lumen also decreased the P _Na /P _Cl. The same effect on P _Na /P _Cl of Ca²⁺ and calcimimetics occurred in ClC-K1 (kidney-specific chloride channel) knockout mice. The addition of 300 μg/ml protamine to the bath strongly inhibited changes to P _Na /P _Cl induced by basolateral Ca²⁺. These data indicate that ambient Ca²⁺ and calcimimetics inhibit Na⁺ transport in the thin ascending limb, which is known to occur via the paracellular shunt pathway. Our observations strongly suggest that Ca²⁺ is involved in the regulation of paracellular Na⁺ permeability in the thin ascending limbs.     

Stunning and myocardial contractile autoregulation studied on the isolated isovolumic blood‐perfused dog heart

Aim: To study, for the first time, the effects of stunning on homeometric and heterometric autoregulation. Methods and results: Ischaemia (15 min)/reperfusion (30 min) was induced in the isovolumic blood‐perfused dog heart preparation. Heart rate elevations (n = 9) from 60 to 200 beats min^?1, in steps of 20 beats min^?1, promoted the same inotropic stimulation in control (C) and stunning (S), indicating that ischaemia/reperfusion does not affect the changes in calcium kinetics elicited by the Bowditch effect. Sudden ventricular dilation (VD) (n = 10) evoked an instantaneous increase in developed pressure (Δ₁DP) followed by a continuous slow performance increase (Δ₂DP) in C and S. Δ₁DP (C: 35 ± 2.2 mmHg; S: 27 ± 2.1 mmHg; P = 0.002) and Δ₂DP (C: 20 ± 1.6 mmHg; S: 14 ± 1.3 mmHg; P = 0.002) decreased proportionally, while Δ₂/Δ₁DP (C: 0.57 ± 0.13; S: 0.58 ± 0.14) and slow response time course (T/2) were unchanged (C: 55 ± 6.6 s; S: 57 ± 7.7 s) after ischaemia/reperfusion. The reduction of Δ₁DP can be understood as a decline of the myofilaments calcium responsiveness, the main pathophysiological effect of stunning. The reason for the weakening of Δ₂DP, due to intracellular calcium gain, was not determined but it was supposed that its complete manifestation could be restricted by cyclic adenosine monophosphate (cAMP) myocardial content reduction. As reported by others, Δ₂DP depends on myocardial cAMP, and it has been shown that myocardial cAMP is decreased after ischaemia/reperfusion. Conclusions: Contractile depression due to stunning has no effect on the inotropic stimulation generated by the Bowditch phenomenon. Immediate and time‐dependent enhancements of contraction evoked by sudden VD are proportionally reduced and the slow response time course is unaffected in the stunned myocardium.     

Gentamicin inhibition of Na+, K+-ATPase in rat kidney cells

Na,K+-ATPase activity is decreased in homogenized renal tissue from GM-treated rats. This study examines whether the site of the active effect of GM on Na,K-ATPase activity in the kidney can be localized to the proximal convoluted tubules (PCT) where the drug is taken up and where it will produce necrosis. In rats treated with gentamicin (50 μg. kg^-1.day^-1 i.m.) for 7 days, PCT Na,K-ATPase activity was reduced as compared to vehicle-treated rats but returned to control levels 7 days after treatment withdrawal. In another nephron segment, the medullary thick ascending limb of Henle (mTAL), where GM induced lesions are uncommon, Na,K-ATPase activity was the same in GM- and vehicle-treated rats treatment. To study the in vitro effect of GM, dissected PCT and mTAL segments from untreated rats were preincubated for 30 min with GM 10^-3m , a dose similar to the tissue concentration in chronically treated rats. In tubule segments that were permeabilized to allow the drug to enter the cells, GM 10^-3m significantly inhibited Na,K-ATPase activity both in PCT and mTAL. In non-permeabilized mTAL segments GM did not inhibit Na,K-ATPase activity. GM inhibition of Na,K-ATPase activity in permeabilized PCT segments persisted after the tubules were rinsed in GM free medium. GM does not inhibit Na,K-ATPase partly purified from the renal cortex. Conclusion. Gentamicin inhibits Na,K-ATPase activity in renal tubule cells when it has access to the cytoplasm. Treatment with GM will therefore cause a selective inhibition of Na,K-ATPase in the proximal tubule cells.     

Body fat and muscle thickness in Japanese and Caucasian females

Fat and muscle thicknesses were measured at eight sites by B-mode ultrasonography on 36 Japanese (age = 25.9 ± 1.9 years; mean ± SD) and 56 Caucasian females (25 ± 2 years) to compare the distribution of these tissues. The eight sites were the biceps, triceps, forearm, subscapular, abdomen, quadriceps, hamstring, and calf. Hydrostatically determined body density, corrected for residual lung volume, was similar (P > 0.05) for Japanese (1.048 ± 0.008 g · ml^?1) and Caucasians (1.050 ± 0.009 g · ml^?1). However, in part because of their greater body mass, Caucasians had significantly more (P < 0.05) fat mass (FM; 12.5 ± 3.2 kg) and fat-free mass (FFM; 45.1 ± 5.1 kg) compared to the Japanese (FM = 11.1 ± 2.3 kg, FFM = 38.0 ± 3.5 kg). From the results of a subsample analysis of a group matched for stature and body mass, Japanese women had a greater abdominal fat thickness than Caucasians, but had less fat thickness at the triceps and hamstring sites. Caucasians had greater muscle thicknesses than Japanese at all sites except for abdomen, hamstring, and calf. The ratio of fat thickness to FM · stature^?2 was higher on the trunk (P < 0.001) in Japanese (6.650 ± 1.721 mm³ · g^?1 × 10³) than in Caucasians (4.713 ± 1.441 mm³ · g^?1 × 10³). The ratio of muscle thickness to FFM · stature² was higher (P < 0.001) in Caucasians than in Japanese at the upper extremity and trunk sites. These results suggest that the distribution of subcutaneous fat might be specific to ethnic origin, and that Japanese women have less muscle development than Caucasians even when matched for stature and body mass. © 1994 Wiley-Liss, Inc.