The role of the angiotensin AT2 receptor on the diurnal variations of nociception and motor coordination in rats.

Phasic pain demonstrates significant diurnal variation in rats. Angiotensin II modulates pain transmission and the diurnal variation in nociception in several rodent pain models. The participation of AT2 receptors in the diurnal regulation of nociception is not yet elucidated. In the present study we investigated the effects of selective peptide AT2 agonist CGP 42112A and the nonpeptide AT2 receptor antagonist PD 123319 on the nociception, motor coordination and arterial blood pressure. Male Wistar 12 weeks old rats were used. CGP 42112A was injected at single doses of 1 and 5 µg/rat intracerebroventricularly (ICV) and infused chronically ICV at a dose of 12 µg/rat/day during 14 days by osmotic minipumps. PD123319 was injected at single doses of 1 and 5 µg/rat, ICV and chronically subcutaneously at a dose of 10 mg/kg/day/14 days. Nociception was assessed by an analgesimeter, arterial blood pressure (ABP) was measured by tail cuff method, and motor coordination by Rota-rod method. Single doses of CGP 42112A (1 and 5 µg/rat) provoked a short lasting antinociception. Unlike acute injection, chronic CGP 42112A infusion increased nociception at the beginning and the end of light phase thus attenuating the diurnal variations observed in the controls. Moreover, it produced an increase of ABP and improved motor coordination. Both acute (1 µg/rat) and chronic PD 123319 treatment resulted in a decrease of pain threshold and chronic treatment attenuated its diurnal fluctuation. Our data support a role for Ang II type 2 receptors in the control of diurnal variations of nociception in rats.

Male obesity and age in relationship to semen parameters and sperm chromatin integrity.

Obesity can adversely affect human health, including fertility. While obesity can disturb the hormonal profile of the female organism and is associated with fertility loss, little is known about what effect male obesity has on fertility. The present study analysed sperm samples of 153 donors. The men were selected from couples attending an infertility clinic, who had tried for 12 months or more to achieve pregnancy without success. The age of the men under investigation was recorded, and their body mass index (BMI) was calculated. All semen samples were assessed for volume, concentration, motility and morphology. Sperm chromatin integrity was measured by sperm chromatin structure assay. Quality of sperm chromatin condensation was assessed by toluidine blue, aniline blue and chromomycin A3 staining. We can conclude that the impact of elevated BMI on the parameters investigated (basic semen parameters, chromatin integrity and chromatin condensation) was not proven in this study. On the other hand, ejaculate quality appeared to be affected by ageing. The impact was reflected by chromatin integrity, which is a factor that can substantially affect fertility in men, rather than by basic sperm parameters.

Effect of the AT1 receptor antagonist losartan on diurnal variation in pain threshold in spontaneously hypertensive rats.

Angiotensin (AT) II plays a key role in the regulation of blood pressure and water-salt balance and modulates nociception. Peptides based on AT influence central functions through the activation of AT1, AT2 or AT4 receptors. The aim of this study was to elucidate the role of AT1 receptors in diurnal variation in nociception in spontaneously hypertensive rats (SHR). Male Wistar rats (16 weeks old) and SHR were caged individually and exposed to light from 08:00 to 20:00 h. The tail cuff method for noninvasive measurement of arterial blood pressure (ABP), paw pressure test for the determination of pain threshold and rotarod test to study motor coordination were used. Chronic treatment was administered to the SHR with the AT1 receptor antagonist losartan (10 mg/kg/day, s.c.) for 14 days. Spontaneously hypertensive rats showed lower pain threshold and smaller day-night variations of nociception as compared to Wistar rats. Chronic losartan decreased the ABP and produced an inverted diurnal pattern of nociception in SHR, increasing the pain threshold at 03:00 h. Neither strain differences nor changes in motor coordination after losartan treatment were observed in SHR. Our results suggest that SHR have disturbances in diurnal variation in nociception and that the AT1 receptor plays a role in the regulation of the circadian rhythm of mechanical pain threshold in SHR.

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples.

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility - idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.

Renal nerves participation in the effects of nitric oxide and ET(A)/ET(B) receptor inhibition in spontaneously hypertensive rats.

The influence of renal nerves on the effects of concurrent NO synthase inhibition (10 mg kg(-1) b.w. i.v. L-NAME) and ET(A)/ET(B) receptor inhibition (10 mg kg(-1) b.w. i.v. bosentan) on renal excretory function and blood pressure in conscious spontaneously hypertensive rats (SHR) was investigated. L-NAME increased blood pressure, urine flow rate, fractional excretion of sodium, chloride and phosphate in both normotensive Wistar rats and SHR with intact renal nerves (p<0.01). GFR or RBF did not change in any of the groups investigated. The effects of L-NAME on renal excretory function were markedly reduced by bosentan and the values returned to control level in the normotensive rats, while in SHR the values were reduced by bosentan, but they remained significantly elevated as compared to control level (p<0.05). The hypertensive response induced by L-NAME in SHR is partially due to activation of endogenous endothelins, but it does not depend on renal nerves. Chronic bilateral renal denervation abolished the effect of L-NAME on sodium and chloride excretion in normotensive rats, whereas it did not alter this effect in SHR. The participation of endogenous endothelins in changes of renal excretory function following NO synthase inhibition is diminished in SHR as compared to Wistar rats.

Renal effects of acute nitric oxide and ET(A)/ET(B) receptor inhibition in conscious spontaneously hypertensive rats.

The aim of the present study was to investigate the effects of endogenous endothelin on renal excretory function in spontaneously hypertensive rats (SHR) after inhibition of NO synthesis. The effects of non-selective ET(A)/ET(B) receptor blockade on L-NAME-induced changes in renal excretory function and blood pressure (BP) were investigated in conscious, SHR and normotensive Wistar rats with implanted catheters in the bladder for urine collection, in the femoral artery for BP registration and in the femoral vein for L-NAME and bosentan administration. L-NAME increased systolic, mean and diastolic BP, diuresis, sodium and chloride excretion (p < 0.01) in both normotensive and hypertensive rats but bosentan returned the values of diuresis, sodium and chloride excretion to control level without any changes in BP in normotensive rats. In SHR the effects of L-NAME were reduced after bosentan (p < 0.05) but the values of diuresis, sodium and chloride excretion still remained statistically significant as compared to control level despite the fact that bosentan lowered mean and diastolic BP increased due to L-NAME administration. Endogenous endothelins participate in a different manner in the rise of BP and in the changes in renal excretory function that develops after L-NAME-induced NO synthase inhibition in normotensive rats and in SHR.

Influence of renal denervation on renal effects of acute nitric oxide and ETA/ETB receptor inhibition in conscious normotensive rats.

The role of renal nerves in the effects of concomitant NO synthase and non-selective ET(A/)ET(B) receptor inhibition on renal function was investigated in conscious normotensive Wistar rats. NO synthase inhibition alone (10 mg/kg b. w. i.v. L-NAME) in sham-operated rats with intact renal nerves induced an increase in systolic, diastolic and mean arterial pressure, urine flow rate, sodium, chloride and calcium excretion (p<0.05). The effect of L-NAME was markedly reduced by bosentan (10 mg/kg b.w. i.v.) and the values of urine flow rate, sodium, chloride and calcium excretions returned to control level (p<0.05). L-NAME administration one week after a bilateral renal denervation increased blood pressure to a similar extent as in sham-operated rats but decreased urine flow rate (p<0.05) and did not change electrolyte excretion. ET(A/)ET(B) receptor inhibition with bosentan during NO synthase inhibition in the renal denervated rats did not produce changes in urine flow rate or electrolyte excretion. NO synthase inhibition as well as concurrent NO synthase and ET(A/)ET(B) receptor inhibition did not change clearance of inulin or paraaminohippuric acid in sham-operated or renal denervated rats. These results indicate that renal sympathetic nerves play an important modulatory role in NO and endothelin induced effects on renal excretory function.

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

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

Influence of nonselective ET(A)/ET(B) receptor blockade on renal function in conscious rats: effects of renal denervation.

The effects of nonselective ET(A)/ET(B) receptor blockade with intravenous bolus injection of bosentan (10 mg/kg) on renal excretory function and blood pressure were investigated in conscious, male, normotensive Wistar rats before and one week after bilateral renal denervation. Renal denervation was followed by an increase in urine flow rate from 4.54+/-0.38 to 5.72+/-0.36 microl/min x 100 g b.w. (p<0.05) and a decrease in urine osmolality from 855.5+/-44.6 to 707.4+/-47.5 mosm/kg H(2)O (p<0.05). Bosentan administration in sham-operated rats resulted in decrease in urine flow rate from 4.54+/-0.38 to 3.49+/-0.34 microl/min x 100 g b.w. (p<0.05), and increase in urine osmolality from 855.5+/-44.6 to 1075.0+/-76.1 mosm/kg H(2)O (p<0.05). Sodium excretion decreased from 226.9+/-20.0 to 155.1+/-11.0 nmol/min x 100 g b.w. (p<0.01). Bosentan administration in renal denervated rats did not produce any changes in renal water or electrolyte excrections. Blood pressure, heart rate, clearance of Inulin or clearance of paraaminohippuric acid (PAH) did not change in sham-operated or renal denervated rats during nonselective ET(A)/ET(B) receptor blockade. Bosentan did not alter the baroreflex sensitivity or sympatho-vagal balance in sham-operated or renal denervated rats. In conclusion, an interaction between renal nerves and endothelins appears to be involved in the regulation of the renal excretory function.

Blood pressure variability in conscious spontaneously hypertensive rats during endothelin A receptor inhibition.

Spontaneously hypertensive rats (SHR) and normotensive Wistar rats were treated with the endothelin(A) (ET(A)) receptor antagonist BQ-123 16.4 nmol/kg.min i.v. at an infusion rate of 25 microl/min for 50 min. Blood pressure was measured in the femoral artery with a Gould/Statham P23ID transducer connected to the computerized data acquisition system Biopac MP100WS. Values of interpulse interval, systolic, diastolic and mean arterial pressure were determined in every heart cycle. Spectrum analysis of systolic, diastolic and mean arterial pressure and interpulse interval variabilities was performed using fast Fourier transform algorithm. In contrast to normotensive rats, BQ-123 infusion in SHR increased mid-frequency band in systolic, diastolic and mean arterial pressure (p < 0.05). The baroreflex sensitivity obtained by the alpha-coefficient in the mid-frequency region (square root of the relation PMFIPI/PMFSAP in msec/mmHg) did not differ between Wistar and SHR before and during BQ-123 administration. Endogenous endothelin acting via endothelin(A) receptors modulates the effects of the sympathetic nervous system on the variations of systolic, diastolic and mean arterial pressure in the hypertensive state. This interrelation between endogenous endothelin and the sympathetic nervous system is not dependent on the arterial baroreflex and is most likely to be realized locally on the vessel level.

Involvement of renal nerves and endothelins in the regulation of renal water excretion in diabetes insipidus rats.

The interaction between renal nerves, endothelins acting via endothelin-A receptors and vasopressin in the regulation of renal excretory function was investigated. In conscious intact and renal denervated diabetes insipidus (DI) Brattleboro rats, as well as their controls, Long-Evans (LE) rats, an infusion of 16.4 nmol/kg/min ET(A) receptor antagonist BQ-123 was performed in the course of 50 min. Femoral artery blood pressure, heart rate, Ccr, V x U(Na), V x U(K) and V x U(Cl) did not alter in any of the groups. Urine flow rate diminished by 38.1% (p < 0.02), while urine osmolality increased by 30.3% (p < 0.05) as a result of BQ-123 infusion in the intact LE rats but neither urine flow rate nor urine osmolality changed in the DI rats. In contrast to intact LE rats, BQ-123 infusion in renal denervated LE rats did not alter urine flow rate or urine osmolality. However, urine flow rate in renal denervated DI rats surprisingly decreased by 71.1% (p < 0.01) while urine osmolality increased by 161% (p < 0.001) as a result of BQ-123 infusion. Endogenous endothelins can regulate renal water excretion through ET(A) receptor activation. Renal sympathetic nerves participate in the modulation of renal water excretion influencing the ET(A) receptor-mediated effects of endothelins in the kidney.

Renal nerves and endothelins interaction in the control of renal excretory function in conscious Long-Evans rats.

The role of renal nerves and endothelins, acting at ET(A) receptors, in the regulation of renal excretory function was investigated in male Long-Evans rats. Catheters were placed in the femoral vein for fluid and drug infusion, in the femoral artery for blood pressure recording as well as in the bladder for urine collection. Infusion of 16.4 nmol/kg/min of the ET(A) receptor antagonist BQ-123 for 50 min was performed in freely moving, intact and renal denervated rats. As a result of BQ- 123 infusion, urine flow rate diminished (P < 0.02) and Uosm increased (P < 0.05) in the intact rats, but not in the renal denervated rats. Bilateral renal denervation itself as well as ET(A) receptor inhibition in both intact and renal denervated rats did not change the mean arterial pressure, heart rate, or the excretion of sodium, potassium and chloride. The data obtained suggest an interrelationship between renal nerves and endothelin-A receptors in the regulation of renal water excretion.

Changes of renal function and blood pressure after nitric oxide synthase inhibition in renal-denervated conscious rats.

Conscious male Wistar rats with implanted catheters in femoral artery for blood pressure registration, femoral vein for drug infusion and bladder for urine collection were used. Nitric oxide synthase inhibition (NOSI) was performed by intravenous administration of 10 mg/kg b.w. N(omega)-nitro-L-arginine methyl ester (L-NAME) before and one week after bilateral renal denervation. Renal denervation led to a decrease of urine osmolality (p<0.05). NOSI decreased heart rate (p<0.001) and increased systolic, mean and diastolic artery pressure both in the intact and the renal-denervated rats (p<0.001). The P(MF)/P(HF) ratio in the heart rate spectrum, considered a criterion for the sympathovagal balance decreased after NOSI in intact and in renal-denervated rats (p<0.001) indicating a reduction in the sympathetic tone. The baroreflex sensitivity after NOSI increased both in conscious rats with intact renal nerves and in rats with bilateral renal denervation. In intact rats NOSI increased urine flow rate by 48.7% (p<0.05), sodium excretion by 339.7% (p<0.01) and chloride excretion by 272.1% (p<0.01). In contrast to the intact rats, NOSI in the renal denervated rats decreased their urine flow rate by 35.5% (p<0.05) and did not alter sodium and chloride excretion. So, chronic renal denervation inversed the diuretic and clearly attenuated the natriuretic and chloruretic response to acute NOSI without inhibiting the rise in blood pressure. Therefore, the increased urine flow as well as the sodium and chloride excretion observed after NOSI are not exclusively the result of a pressure diuresis but are somewhat dependent on the renal sympathetic nerve activity. Renal denervation did not change the pattern of sympathovagal balance and baroreflex sensitivity modified by NO-synthase inhibition.

Spectral coherence between blood pressure and inter-beat intervals in hypertension.

Continuous registration of blood pressure (BP) was obtained in 31 normotensives and 76 hypertensives (divided into 3 stages of hypertension) by a method where the blood pressure in a human finger was measured non-invasively in a beat-by-beat fully calibrated manner. From this signal, the power density spectra of inter-beat intervals (IBI), systolic blood pressure (SBP) and diastolic blood pressure (DBP) were derived. Total spectral power (TP) and low/high spectral frequency ratios of all spectra were calculated. Cross-correlation analysis between IBI, SBP and DBP spectra showed that the level of amplitude and phase spectral coherence (SC) was strongly dependent of the stage of hypertension. SC was calculated by means of appropriate mathematical algorithms. Multiple step regression analysis pointed out that the observed phenomenon is highly significant for both--the amplitude and the phase SC (p < 0.01).

Renal excretory function in conscious Long Evans and vasopressin deficient (Brattleboro) rats after endothelin-A receptor inhibition.

All experiments were performed on conscious, freely moving male Long Evans as well as Diabetes incipidus (Brattleboro) rats (300-320 g). The endothelin-A (ETA) receptor antagonist BQ-123 (Neosystem) was administered through femoral vein cannula. Arterial blood pressure was measured trough femoral artery catheter. The bladder was cannulated for urine collection via a small suprapubic incision. After a 40 min control period BQ-123 infusion (16.4 nmol/kg/min, 25 microliters/min) was started and continued for 50 min. The effect of 32.8 nmol/kg/min BQ-123 infused in conscious Brattleboro rats was also investigated. Plasma and urine concentrations of sodium, potassium and chloride as well as osmolality were determined. Glomerular filtration rate (GFR) was estimated using the clearance of endogenous creatinine. Endothelin-A receptor inhibition by 16.4 nmol/kg/min BQ-123 infusion in conscious Long-Evans rats decreased urine flow rate by 38.4% (p < 0.02) and increased urine osmolality by 30.3% (p < 0.05). Sodium, potassium, chloride excretion did not alter. Endothelin-A receptor inhibition by 16.4 nmol/kg/min and by 32.8 nmol/kg/min BQ-123 infusion in conscious Brattleboro rats did not produce any change in urine flow rate, urine osmolality or excretion of the electrolytes studied. Endothelins acting via ETA receptors may function as an inhibitor of water reabsorption in the kidneys of conscious rats.

Spectral analysis of heart rate and arterial pressure variability after nitric oxide synthase inhibition.

The experiments were performed on male, conscious Wistar rats. Femoral arterial pressure was registered by Statham GOULD P23 ID pressure transducer connected to MP 100WS BIOPAC work station after analog to digital conversion during 40 minutes long control period. Nitric oxide synthase inhibition was performed by injection of 100 microliters, 10 mg/kg b.w. N-omega-nitro-L-arginine methyl ester (L-NAME) in saline through femoral vein catheter. Twenty minutes later arterial pressure registration was started and was continued for 40 minutes. The pulse-by-pulse values of systolic, diastolic and mean arterial pressure as well as the pulse intervals were measured by peak and rate detectors of the AcqKnowledge 2.0 software. Row data were processed using a virtual instrument developed in our laboratory in the graphical programming environment Lab VIEW 3.1.1. L-NAME increased systolic, diastolic and mean arterial pressure by 16.6%, 25% and 35%, respectively. The PMF/PHF ratio in heart rate spectrum decreased, indicating an increased vagal effect on the heart. Nitric oxide synthase inhibition increased the low-frequency component of systolic arterial blood pressure variability by 39.5%. Nitric oxide is a physiological regulator of rapid fluctuations of arterial blood pressure.