Mariette Barthelmebs

Paradoxical actions of exogenous and endogenous parathyroid hormone-related protein on renal vascular smooth muscle cell proliferation: reversion in the SHR model of genetic hypertension

In previous studies, added parathyroid hormone‐related protein (PTHrP) inhibits whereas transfected PTHrP stimulates the proliferation of A10 aortic smooth muscle cells by nuclear translocation of the peptide. In the present studies, we asked whether these paradoxical trophic actions of PTHrP occur in smooth muscle cells (SMC) cultured from small intrarenal arteries of, and whether they are altered in, 12‐wk‐old spontaneously hypertensive rats (SHR) as compared to normotensive Wistar‐Kyoto (WKY) rats. SHR cells grew faster than WKY cells. PTHrP transcript was increased in SHR‐derived cells whereas PTH1 receptor (PTH1R) transcripts were similar in both cell lines. In both strains of cells, stable transfection with human PTHrP(1–139) cDNA did not further induce proliferation, suggesting maximal effect of endoge‐nous PTHrP in wild cells. In contrast, transfection with antisense hPTHrP(1–139) cDNA, which abolished PTHrP mRNA, decreased WKY but increased SHR cell proliferation. Added PTHrP(1–36) (1–100 pM) de¬creased WKY and increased SHR cell proliferation. Additional studies indicated that the preferential cou¬pling of PTH1‐R to G‐protein Gi was responsible for the proliferative effect of exogenous PTHrP in SHR cells. Moreover, PTHrP was detected in the nucleolus of a fraction of WKY and SHR renal SMC, in vitro as well as in situ, suggesting that the nucleolar transloca¬tion of PTHrP might be involved in the proliferative effects of endogenous PTHrP. In renovascular SMC, added PTHrP is antimitogenic, whereas endogenously produced PTHrP is mitogenic. These paradoxical ef¬fects of PTHrP on renovascular SMC proliferation appear to be reversed in the SHR model of genetic hypertension. A new concept emerges from these re¬sults, according to which a single molecule may have opposite effects on VSMC proliferation under physio¬logical and pathophysiological conditions.—Mass¬felder, T., Taesch, N., Endlich, N., Eichinger, A., Escande, B., Endlich, K., Barthelmebs, M., Helwig, J.‐J. Paradoxical actions of exogenous and endogenous parathyroid hormone‐related protein on renal vascular smooth muscle cell proliferation: reversion in the SHR model of genetic hypertension. FASEB J. 15, 707‐718 (2001)

Signal transduction pathways involved in kinin B2 receptor‐mediated vasodilation in the rat isolated perfused kidney

The signal transduction pathways involved in kinin B2 receptor‐related vasodilation were investigated in rat isolated perfused kidneys. During prostaglandin F2α or KCl‐induced constriction, the vasodilator response to a selective B2 receptor agonist, Tyr(Me)8bradykinin (Tyr(Me)8BK), was assessed. Tyr(Me)8BK produced a concentration‐ and endothelium‐dependent relaxation that was decreased by about 30 – 40% after inhibition of nitric oxide (NO) synthase by NG‐nitro‐L‐arginine (L‐NOARG) or of cyclo‐oxygenase by indomethacin; a greater decrease (about 40 – 50%) was observed after concomitant inhibition of the two pathways. High extracellular K+ diminished Tyr(Me)8BK‐induced relaxation by about 75% suggesting a major contribution of endothelium‐derived hyperpolarization. The residual response was almost completely suppressed by NO synthase and cyclo‐oxygenase inhibition. The K+ channel inhibitors, tetrabutylammonium (non‐specific) and charybdotoxin (specific for Ca2+‐activated K+ channel), suppressed Tyr(Me)8BK‐induced relaxation resistant to L‐NOARG and indomethacin. Inhibition of cytochrome P450 (clotrimazole or 7‐ethoxyresorufin) decreased the NO/prostanoids‐independent relaxation to Tyr(Me)8BK by more than 60%, while inhibition of the cannabinoid CB1 receptor (SR 141716A) had only a moderate effect. Acetylcholine induced a concentration‐dependent relaxation with characteristics nearly similar to the response to Tyr(Me)8BK. In contrast, the relaxation elicited by sodium nitroprusside was potentiated in the absence of NO (L‐NOARG or removal of endothelium) but remained unchanged otherwise. These results indicate that the activation of kinin B2 receptors in the rat isolated kidney elicits an endothelium‐dependent vasorelaxation, mainly dependent on the activation of charybdotoxin‐sensitive Ca2+‐activated K+ channels. In addition, cytochrome P450 derivatives appear to be involved.

Vascular kinin B1 and B2 receptor-mediated effects in the rat isolated perfused kidney–differential regulations

Bradykinin (BK) and analogs acting preferentially at kinin B1 or B2 receptors were tested on the rat isolated perfused kidney. Kidneys were perfused in an open circuit with Tyrodes solution. Kidneys preconstricted with prostaglandin F2α were used for the analysis of vasodilator responses. BK induced a concentration‐dependent renal relaxation (pD2=8.9±0.4); this vasodilator response was reproduced by a selective B2 receptor agonist, Tyr(Me)8‐BK (pD2=9.0±0.1) with a higher maximum effect (Emax=78.9±6.6 and 55.8±4.3% of ACh‐induced relaxation respectively, n=6 and 19, P<0.02). Icatibant (10 nM), a selective B2 receptor antagonist, abolished BK‐elicited relaxation. Tachyphylaxis of kinin B2 receptors appeared when repeatedly stimulated at 10 min intervals. Des‐Arg9‐BK, a selective B1 receptor agonist, induced concentration‐dependent vasoconstriction at micromolar concentration. Maximum response was enhanced in the presence of lisinopril (1 μM) and inhibited by R 715 (8 μM), a selective B1 receptor antagonist. Des‐Arg9‐[Leu8]‐BK behaved as an agonist. A contractile response to des‐Arg9‐BK occurred after 1 h of perfusion and increased with time by a factor of about three over a 3 h perfusion. This post‐isolation sensitization to des‐Arg9‐BK was abolished by dexamethasone (DEX, 30 mg kg−1 i.p., 3 h before the start of the experiment and 10 μM in perfusate) and actinomycin D (2 μM). Acute exposure to DEX (10 μM) had no effect on sensitized des‐Arg9‐BK response, in contrast to indomethacin (30 μM) that abolished it. DEX pretreatment however had no effect on BK‐induced renal vasodilation. Present results indicate that the main renal vascular response to BK consists of relaxation linked to the activation of kinin B2 receptors which rapidly desensitize. Renal B1 receptors are also present and are time‐dependently sensitized during the in vitro perfusion of the rat kidneys.

AT2-antagonist sensitive potentiation of angiotensin II-induced vasoconstrictions by blockade of nitric oxide synthesis in rat renal vasculature.

1 Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT1 receptor subtype, AT2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal effects of vasoconstrictor drugs, we queried whether the Ang II‐induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the effect of AT1 and AT2 receptor (AT‐R) antagonists on Ang II‐induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant flow in a single‐pass circuit. 2 In the absence of NO blockade, the AT1‐R antagonist L‐158809 (500 nM) antagonized the Ang II‐induced vasoconstrictions, while the AT2‐R antagonist PD‐123319 (500 nM) had no effect. 3 Perfusing kidneys in the presence of either NO synthase inhibitors, L‐NAME (100 μM) or L‐NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY‐83583 (10 μM), significantly increased both molar pD2 (from 9.40±0.25 to 10.36±0.11) and Emax values (from 24.9±3.1 to 79.9±4.9 mmHg) of the concentration–response curve for Ang II‐induced vasoconstriction. 4 In the presence of L‐NAME, 500 nM L158809 abolished the Ang II‐induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD‐123319 reversed the left shift of the concentration–response curve for Ang II (molar pD2 value 9.72±0.13) leaving Emax value unaffected (91.3±7.6 mmHg). 5 In the presence of L‐NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration‐dependent manner by L‐158809 (0.05–500 nM). By contrast, PD‐123319 (0.5–500 nM) did not affect the 10 nM Ang II‐induced vasoconstriction and concentration‐dependently decreased the 0.1 nM Ang II‐induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist. 6 Similar to PD‐123319, during NO blockade the AT2‐R antagonist CGP‐42112A at 5 nM decreased by 50% the 0.1 nM Ang II‐induced vasoconstriction and at 500 nM had no effect on 10 nM Ang II‐induced vasoconstriction. 7 In conclusion, the renal Ang II‐induced vasoconstriction, which is antagonized only by AT1‐R antagonist in the presence of endogenous NO, becomes sensitive to both AT1‐ and AT2‐R antagonists during NO synthesis inhibition. While AT1‐R antagonist inhibited both L‐NAME‐potentiated and‐augmented components of Ang II‐induced vasoconstriction, AT2‐R antagonists inhibited only the L‐NAME‐potentiated component.

Role of shear stress in nitric oxide-dependent modulation of renal angiotensin II vasoconstriction.

Renal vasoconstriction in response to angiotensin II (ANGII) is known to be modulated by nitric oxide (NO). Since shear stress stimulates the release of a variety of vasoactive compounds from endothelial cells, we studied the impact of shear stress on the haemodynamic effect of ANGII in isolated perfused kidneys of rats under control conditions and during NO synthase inhibition with L‐NAME (100 μM). Kidneys were perfused in the presence of cyclo‐oxygenase inhibitor (10 μM indomethacin) with Tyrodes solution of relative viscosity ζ=1 (low viscosity perfusate, LVP) or, in order to augment shear stress, with Tyrodes solution containing 7% Ficoll 70 of relative viscosity ζ=2 (high viscosity perfusate, HVP). Vascular conductance was 3.5±0.4 fold larger in HVP as compared with LVP kidneys, associated with an augmentation of overall wall shear stress by 37±5%. During NO inhibition, vascular conductance was only 2.5±0.2 fold elevated in HVP vs LVP kidneys, demonstrating shear stress‐induced vasodilatation by NO and non‐NO/non‐prostanoid compound(s). ANGII (10–100 pM) constricted the vasculature in LVP kidneys, but was without effect in HVP kidneys. During NO inhibition, in contrast, ANGII vasoconstriction was potentiated in HVP as compared with LVP kidneys. The potentiation of ANGII vasoconstriction during NO inhibition has been shown to be mediated by endothelium‐derived P450 metabolites and to be sensitive to AT2 receptor blockade in our earlier studies. Accordingly, in HVP kidneys, increasing concentrations of the AT2 receptor antagonist PD123319 (5 and 500 nM) gradually abolished the potentiation of ANGII vasoconstriction during NO inhibition, but did not affect vasoconstriction in response to ANGII in LVP kidneys. Our results demonstrate, that augmentation of shear stress by increasing perfusate viscosity induces vasodilatation in the rat kidney, which is partially mediated by NO. Elevated levels of shear stress attenuate renal ANGII vasoconstriction through enhanced NO production and are required for AT2 sensitive potentiation during NO inhibition.

Diabetes-induced changes in the 5-hydroxytryptamine inhibitory receptors involved in the pressor effect elicited by sympathetic stimulation in the pithed rat

1 We investigated the effect of alloxan‐induced diabetes on the inhibitory mechanisms of 5‐hydroxytryptamine (5‐HT) in the pressor responses induced by stimulation of sympathetic vasopressor outflow in pithed rats, and analysed the type and/or subtype of 5‐HT receptors involved. 2 Diabetes was induced in male Wistar rats by a single s.c. injection of alloxan, then 4 weeks later, they were anaesthetized, pretreated with atropine and pithed. Electrical stimulation of the sympathetic outflow from the spinal cord (0.1, 0.5, 1 and 5 Hz) resulted in frequency‐dependent increases in blood pressure. 3 Intravenous infusions of 5‐HT (1–80 μg kg−1 min−1) reduced the pressor effects obtained by electrical stimulation. The 5‐HT1 receptor agonist 5‐carboxamidotryptamine, 5‐CT (5 μg kg−1 min−1), caused an inhibition of the pressor response, whereas the selective 5‐HT2 receptor agonist, α‐methyl‐5‐HT (5 μg kg−1 min−1) and the selective 5‐HT3 receptor agonist, 1‐phenylbiguanide (40 μg kg−1 min−1), did not modify the sympathetic pressor responses. 5‐HT had no effect on exogenous noradrenaline (NA)‐induced pressor responses. 4 The inhibition of electrically induced pressor responses by 5‐HT (10 μg kg−1 min−1) was unable to be elicited after i.v. treatment with methiothepin (100 μg kg−1) because of the marked inhibition produced by methiothepin alone. The 5‐HT‐induced inhibition was blocked after i.v. administration of WAY‐100,635 (100 μg kg−1) and not affected by ritanserin (1 mg kg−1), MDL 72222 (2 mg kg−1). 5 The selective 5‐HT1A receptor agonist, 8‐hydroxydipropylaminotretalin hydrobromide (8‐OH‐DPAT) (5–20 μg kg−1 min−1) but neither the rodent 5‐HT1B receptor agonist, CGS‐12066B (5 μg kg−1 min−1), nor the selective nonrodent 5‐HT1B and 5‐HT1D receptor agonist, L‐694,247 (5 and 40 μg kg−1 min−1), inhibited the electrically induced pressor response. The selective 5‐HT1A receptor antagonist, WAY‐100,635 (100 μg kg−1), blocked the inhibition induced by 8‐OH‐DPAT (10 μg kg−1 min−1). 8‐OH‐DPAT had no effect on exogenous NA‐induced pressor responses. 6 Experimental diabetes produces changes in the inhibitory effect induced by 5‐HT on electrically induced sympathetic pressor responses, such that the inhibitory action induced by 5‐HT in diabetic pithed rats is mediated by prejunctional 5‐HT1A receptors.

Vascular catabolism of bradykinin in the isolated perfused rat kidney.

Kinins in the circulation are rapidly metabolized by several different peptidases. The purpose of this study was to evaluate the contribution of membrane-bound peptidases to kinin metabolism in the renal circulation. Experiments were performed in vitro, in isolated rat kidneys perfused at a constant flow rate (8 ml/min) with Tyrodes solution. The effects of peptidase inhibitors were evaluated on the functional vasodilator response caused by bradykinin (30 nM) or [Tyr(Me)(8)]bradykinin (10 nM) via activation of bradykinin B2 receptors in kidneys precontracted with prostaglandin F2alpha. Angiotensin converting enzyme inhibitors, enalaprilat (3 microM), ramiprilat (1 microM) or lisinopril (1 microM), increased the bradykinin-induced renal vasodilation by 40% or more. Inhibitors of neutral endopeptidase (thiorphan or phosphoramidon, 10 microM), basic carboxypeptidase (DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid or MGTPA, 10 microM) and aminopeptidase P (apstatin, 20 microM) however did not enhance the renal vasodilator response elicited by kinins, whatever tested alone or in the presence of lisinopril. These findings indicate that angiotensin converting enzyme is the major peptidase whose inhibition potentiates the renal bradykinin B2 receptor mediated vasodilator response of kinins. The relative contribution in this potentiation of inhibition of kinin inactivation and of cross-talk of angiotensin converting enzyme with bradykinin B2 receptor remains however to be clarified.

Oxytocin-induced renin secretion by denervated kidney in anaesthetized rat

The effects of oxytocin on renin secretion by denervated kidney were investigated in vivo, by infusing the peptide directly into the renal artery of anaesthetized rats. Renin secretion was calculated by the renal veno-arterial difference in plasma renin activity multiplied by renal plasma flow. The intra-renal arterial (i.r.a.) infusion of oxytocin (1.5 or 15 ng/kg/min, 10 min) induced a sixfold increase in renin secretion as compared to vehicle-treated controls, without effects on renal blood flow, mean arterial blood pressure, glomerular filtration rate or natriuresis. The effect of oxytocin (1.5 ng/kg/min) was prevented by pretreatment with an oxytocin receptor antagonist, desGly-NH(2),d(CH(2))(5)[D-Tyr(2),Thr(4),Orn(8)]vasotocin] (5.6 microg/kg bolus i.v. 20 min before oxytocin infusion, followed by 2.8 microg/kg/min i.r.a.). Nadolol (2.5 mg/kg i.v.), a beta-adrenoceptor antagonist, also blocked the oxytocin-induced increase in renin secretion. These results show that oxytocin is able to stimulate renin release by activating oxytocin receptors but that beta-adrenoceptors also seem to be involved.

Renal vascular reactivity to vasopressin in rats with diabetes mellitus

We evaluated how renal vascular reactivity to vasopressin changes when nitric oxide (NO) synthesis varies, as has been reported to occur in the course of insulin-dependent diabetes mellitus. Renal vasoconstrictor responses to vasopressin were obtained in young and older Sprague-Dawley control rats (3 and 10 months old) and in age-matched diabetic rats that had been treated with streptozotocin (60 mg/kg i.v.) at the age of 2 months. In young rats, vasopressin (3-1000 ng/kg/min i.v.) induced in vivo a dose-dependent decrease in renal blood flow, which was diminished in streptozotocin diabetic rats (P<0.05). Similarly, in in vitro perfused kidneys, the concentration-response curve for vasopressin (0.03-10 nM) was shifted 3-fold to the right in kidneys isolated from young diabetic rats (P<0.05). This shift was abolished in the presence of an inhibitor of nitric oxide synthesis, N(G)-nitro-L-arginine (100 microM), in the perfusate. In 10-month-old rats, the in vivo renal vasoconstrictor dose-response curve to vasopressin was shifted 10-fold to the left as compared to that for young rats (P<0.001). This shift was similar in both control and diabetic rats. In conclusion, the present study documented the existence of hyporesponsiveness to vasopressin in the early stage of diabetes, possibly related to nitric oxide overproduction. In contrast, renal vascular hyperreactivity to vasopressin occurs with aging, whether the rats are diabetic or not.