Nitric oxide synthase activity and gender influence in pulmonary hypertension

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POSTERS: Experimental Hypertension

used as a marker of hydroxyl radical generation and was measured in dialysis samples by HPLC with electrochemical detection. Some quantity of 2,3-DHBA was always presented in the perfusion fluid due to exogenous salicylic acid oxidation. This value in each experiment was considered as 100% for the following calculations. Mean baseline levels of 2,3-DHBA in the myocardial dialysate samples of WKY rats were not different (112⫾8,7%, n⫽5) from that in perfusion fluid, but were significantly lower (87⫾3.2%, n⫽ 6, p⬍0.03) in Wistar rats and significantly higher (154⫾1.1%, n⫽7, p⬍0.0001) in SHR-SP. We assume that this is the first report of in vivo registration of the elevated basal hydroxyl radical generation in hypertrophied miocardium of SHR-SR by high sensitive salicylate trapping combined with microdialysis technique. Moreover, basal hydroxyl radical level in SHR-SP was strongly correlated (r⫽0.831, n⫽7, p⬍0.05) with degree of LV hypertrophy expressed as a ratio of LV weight to the body weight. These results allow us to suggest that LV hypertrophy in SHR-SP is accompanied by the elevated hydroxyl radical production. Key Words: Myocardium Hypertrophy, Hydroxyl Radical, Microdialysis with Salicylate

P-325 ENHANCED JNK2 ACTIVITY IN THE HYPERTROPHIC HEART VENTRICLE OF PRAGUEHYPERTENSIVE RAT (PHR) IS INDEPENDENT OF CIRCULATING OR TISSUE ENDOTHELIN (ET) Herbert J. Kramer, Angela Baecker, Dirk Bokemeyer, Volker Vogel, Jiri Heller. Renal Section, Medical Policlinic, University of Bonn, Germany; Exp. Medicine, Inst. Clin. Exp. Med., Prague, Czech Republic. Since MAP kinases may be involved in cardiac hypertrophy in spontaneously hypertensive rats and their activation may be due to stimulation by ET, we studied MAP kinases ERK, JNK and p38 and gene expression of preproET-1 mRNA and of ECE-1 mRNA in atrial and ventricular tissues of PHR and their normotensive controls (PNR). Systolic blood pressure was 208⫾15 in PHR vs 121⫾5 mm Hg in PNR (p⬍0.01). Total heart weight was 316⫾4 in PHR vs 247⫾4 mg/100 g b.w. in PNR (p⬍0.01). Left and right ventricular weights were 168⫾4 (p⬍0.01) and 57⫾2 (n.s.) in PHR vs 121⫾5 and 53⫾3 mg/100 g b.w. in PNR. Western blot analysis and immunocomplex activity assay showed no differences in ERK or p38 MAP kinase but significantly greater phosphorylation of JNK2 in PHR than in PNR with an increase in JNK2 activity in left ventricle to 6.4⫾1.5 in PHR vs 2.5⫾0.5 O.D. in PNR (each n⫽5; p⬍0.05). Plasma ET-1,2 was 10.5⫾1.9 in PHR vs 12.3⫾1.3 fmol/ml in PNR (each n⫽12). Using competitive PCR for quantitative analysis preproET-1 mRNA in heart ventricle was 1.20⫾0.27 in PHR vs 1.10⫾0.22 fmol/mg RNAtotal in PNR. In atrial tissue it was 2.68⫾0.49 in PHR vs 2.91⫾1.04 fmol/mg RNAtotal in PNR (each n⫽5; n.s.). We found between PHR and PNR no differences in ECE-1 mRNA expression in heart ventricle (0.68⫾0.15 vs 0.64⫾0.11 fmol/mg RNAtotal) or in heart atrium (1.10⫾0.51 vs 1.11⫾0.38 fmol/mg RNAtotal). Similarly, no differences in cardiac ET-1 protein content were noted. Thus, despite marked cardiac hypertrophy atrial and ventricular preproET-1 mRNA and ECE-1 mRNA expression was similar in PHR and PNR suggesting that the rise in JNK2 activity, probably contributing to ventricular hypertrophy in this model of spontaneous hypertension, was independent of the circulating or local tissue ET-1 system. Key Words: Endothelin, MAP-Kinase, Cardiac Hypertrophie

AJH–April 2002–VOL. 15, NO. 4, PART 2

P-326 ASSOCIATION BETWEEN A DEFICIENT CARDIOTROPHIN-1 SIGNALING PATHWAY AND CARDIAC APOPTOSIS IN GENETIC HYPERTENSION Susana Ravassa, Noelia Ardanaz, Paula Mun˜ iz, Arantxa Gonzalez, Begon˜ a Lo´ pez, Javier Dı´ez, Marı´a A. Fortun˜ o. Division of Cardiovascular Pathophysiology, School of Medicine, University of Navarra, Pamplona, Spain. The present study was designed to investigate whether cardiotrophin-1 (CT-1), and its signaling molecules gp130 and the leukemia inhibitory factor receptor (LIF-R) participate in the cardiac hypertrophy and increased cardiomyocyte apoptosis present in adult spontaneously hypertensive rats (SHR). We also studied the effects of a calcium channel antagonist on these cardiac abnormalities. Twenty 16-weeks old SHR were treated with 1 (n⫽10) or 7.5 (n⫽10) mg/Kg/day of lercanidipine during 14 weeks. Ten normotensive Wistar Kyoto (WKY) rats, ten untreated SHR and the lercanidipine treated SHR were sacrificed and hearts were extracted. The expression of CT-1, gp130 and LIF-R was quantified by Western blotting, apoptosis was measured by TdT reaction and cardiomyocyte size was determined by quantitative morphometry using an image analysis system. Compared with WKY, untreated SHR exhibited both gp130 and LIF-R expression decreased (P⬍0.05 and P⬍0.01, respectively) whereas the amount of the CT-1 was similar in hearts from the two groups of rats. Moreover, untreated SHR showed increased blood pressure (P⬍0.01), increased cardiac index and cardiomyocyte size (P⬍0.01), and increased apoptotic index (P⬍0.05) as compared to WKY. Only at high doses, lercanidipine significantly augmented (P⬍0.01) CT-1 expression, decreased (P⬍0.01) blood pressure, cardiac index (P⬍0.05), transversal diameter of cardiomyocytes (P⬍0.01) without modifying longitudinal diameter and completely diminished apoptotic index (P⬍0.01). gp130 and LIF-R were not affected by treatment. Our data show that a diminished expression of gp130 and LIF-R proteins is associated with cardiomyocyte apoptosis in SHR. The increase in CT-1 expression after lercanidipine treatment is associated with the normalization of apoptosis. Thus, we suggest that a diminished CT-1/gp-130-LIF-R pathway may contribute to the excessive cardiac apoptosis that takes place in the heart of adult SHR. Key Words: Cardiotrophin 1, Apoptosis, Hypertension

P-327 NITRIC OXIDE SYNTHASE ACTIVITY AND GENDER INFLUENCE IN PULMONARY HYPERTENSION Palaniswamy Vijay, Thomas G. Sharp, Sameer Khatri, Yechang Ge, John W. Sharp. Surgery, Indiana University School of Medicine, Indianapolis, IN, United States. Pulmonary hypertension (PH) is a progressive disease that leads to right heart failure and death. Various vasoactive mediators play an important role in the disease progression. In this study we have analyzed changes in nitric oxide synthase(NOS) activity and adrenomedullin (ADM)-a vasodilator, and as well as the influence of gender on the development of PH. Male/Female SD rats were injected subcutaneously with a single dose of 60mg/kg b wt monocrotaline (MCT). Control rats received the solvent alone. Rats were terminated 7 and 21 days after treatment and blood ADM levels (pg/ml) and lung NOS activity (pmol/min/g protein) were measured. The mortality rates were 33% and 3% for male and female rats respectively. ADM levels and NOS activity were higher in female rats when compared to male rats as shown in the table. NOS activity increased initially 2-fold in male MCT rats and then declined sharply from 7 to 21 days (p⬍0.001).

AJH–April 2002–VOL. 15, NO. 4, PART 2

POSTERS: Experimental Hypertension

MCT is more toxic in male rats, but a delayed effect was seen in females. In female rats the NOS induction is delayed but lasts longer to protect against the effect of MCT. ADM levels in female rats are higher than in male rats. The increased levels of vasodilatory peptides can protect female rats from pulmonary hypertension thereby decreasing the mortality rate.

Male

NOS NOS ADM ADM

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reflex excitatory effect upon stimulation of renal receptors or of nonrenal sympathetic afferents, or, more probably, from central and ganglionic excitation of sympathetic neurons by a substance released by the ischaemic kidney. Key Words: Renal Nerve Activity, Functional Nephrectomy, Renin-Angiotensin System

Female

Time (days)

Control (n ⴝ 7)

MCT (n ⴝ 7)

Control (n ⴝ 7)

MCT (n ⴝ 7)

7 21 7 21

32 ⫾ 0.6 38 ⫾ 1.1 2.6 ⫾ 0.6 4.7 ⫾ 0.8

67 ⫾ 0.8* 34 ⫾ 0.7* 3.4 ⫾ 0.8 6.2 ⫾ 0.4

38 ⫾ 0.5 47 ⫾ 0.4 2.3 ⫾ 0.5 5.4 ⫾ 1.7

52 ⫾ 0.7 58 ⫾ 0.8 4.1 ⫾ 0.6 9.5 ⫾ 2.4

Key Words: Pulmonary Hypertension, NOS, Adrenomedullin

P-329 THREE CHROMOSOMAL REGIONS MODULATE AN IMPAIRED ENDOTHELIAL DEPENDENT VASORELAXATION IN THE STROKE PRONE SPONTANEOUSLY HYPERTENSIVE RAT P-328 THE EFFECTS OF EXPERIMENTAL ACUTE RENAL HYPERTENSION ON SYMPATHETIC NERVE ACTIVITY TO THE KIDNEY IN THE RAT Giorgio Recordati, Federica Zorzoli, Olivia Pontara, Alberto Zanchetti. Centro Fisiologia Clinica ed Ipertensione, University of Milan, Milano, Italy. The reopening of the renal hilum after a period of complete renal ischaemia is followed by a hypertensive crisis (the experimental acute renal hypertension: EARH) which is due to the acute release of renin and endogenous angiotensin II formation. The present study was done to verify if the EARH is accompanied by simultaneous alterations in efferent sympathetic nerve activity to the kidney. The experiments were done on Sprague-Dawley spontaneously breathing rats (200-300 g) anaesthetized with nembutal (n⫽5). After excision of the left kidney, the renal nerves to the right kidney were exposed by a retroperitoneal approach and a single nerve strand was positioned on a bipolar hook electrode to record renal sympathetic postaganglionic nerve activity (RSNA). Threads were placed around the right renal artery and vein in order to produce a complete renal ischaemia. After three hours these ties could be loosened to reestablish blood flow to and from the kidney (functional right nephrectomy: FRN). RSNA, blood pressure, heart rate, rate of breathing and rectal temperature were continuously recorded. From the beginning of the experiment (time 0) to its end (time 300 min) the nerve strands were left untouched and their position on the electrode was unchanged. As previously reported with respect to control conditions RSNA decreased during three hours of FRN from 39.1 ⫾3 imp/sec at baseline, to 13.7 ⫾3 at the end of three hours of FRN period (Recordati, J Hypertens, 18: 1277, 2000). The present report describes what occurred at the reopening of the right renal hilum when a hypertensive crisis and tachycardia are observed. In four out of five rats this hypertension was accompanied by a 1-3 min continuous firing of the RSNA to a mean peak increase of 28.7 ⫾7 imp/sec at a time the mean systolic blood pressure had increased to 209.4 ⫾21 mmHg. RSNA subsequently recovered towards a spontaneous normal bursting discharge, while blood pressure persisted elevated. These results indicate that the increase in sympathetic activity at the reopening of the renal hilum is short-lasting and therefore may only be partially responsible for the observed persistent increase in blood pressure. The consistently observed excitation of RSNA may be due to a

Bruna Gigante, Speranza Rubattu, Rosita Stanzione, Alessia Lombardi, David Tarasi, Paola Spinsanti, Massimo Volpe. Istituto Neurologico Mediterrraneo ‘Neuromed‘, Pozzilli (Is), Italy; Dept of Pathology and Experimental Medicine, La Sapienza University, Rome, Italy. An impaired endothelial-dependent vasorelaxation (EDV) precedes cerebrovascular lesions in an experimental animal model of inherited stroke, the stroke prone spontaneously hypertensive rat (SHRSP). Experiments performed in our laboratory suggest a genetic predisposition to EDV in this experimental model that segregates in the SHRSP/SHR F2 generation independently from blood pressure. The aim of the present study was to investigate the effects of genetic factors on EDV performing a random marker genome screening. One-hundred thirty-seven F2 (64 male, 73 female) rats 6week-old and the parental strains were studied. Systolic blood pressure (SBP) was recorded and the vasodilator response to acetilcholine on rings from thoracic aorta pre-constricted with phenylephrine was assessed. The segregation of the fractional vasorelaxation (D ratio) in the F2 was chosen as quantitative phenotype. One-hundred microsatellites from a panel of markers polymorphic between SHR and SHRSP, spaced on average every 15 KcM along the rat genome, were tested. Data were expressed as mean⫾SEM; for each genotype marker one-way analysis of variance was carried out. Pearson test was used to assess a correlation between SBP and D ratio in the F2. SBP (mmHg) was not different among the three groups (178⫾7 SHR, 180⫾6 SHRSP, 182⫾7 F2) and no correlation was found with D ratio (r⫽0.08, p⫽0.48); D ratio was reduced in the SHRSP compared to SHR (0.45⫾0.02 vs 0.75⫾0.02, p⬍0.05) and 0.64⫾0.03 in the F2 population, therefore the phenotypic variance genetically determined in this model is 94%. We have identified three markers located on chromosome 1, D1Wox4 (D ratio 0.71⫾0.03 SHR/SHR, 0.62⫾0.03 SP/SP, 0.58⫾0.03 SHR/SP, F2,119⫽3.7 p⫽0.027), chromosome 7, D7Wox4 (D ratio 0.71⫾0.04 SHR/SHR, 0.57⫾0.03 SP/SP, 0.60⫾0.03 SHR/SP, F2,119⫽3.5 p⫽0.032) and chromosome 14, D14Rat58 (D ratio 0.72⫾0.03 SHR/SHR, 0.54⫾0.04 SP/SP, 0.65⫾0.03 SHR/SP, F2, 82⫽3.8 p⫽0.026) influencing the D ratio with the SHR allele conferring a protective effect. Our results strongly support a genetic predisposition to EDV in this experimental model and indicate that is influenced by at least three different genetic loci each one exerting a weak effect on the phenotype. Key Words: Endothelial Dysfunction, Genetics, Intermediate Phenotypes