Domenico Palomba

Cardiovascular Phenotype of a Mouse Strain With Disruption of Bradykinin B2-Receptor Gene

BACKGROUND To evaluate the role of kinins in the regulation of cardiovascular function, we studied the phenotype of a mouse strain with disruption of the bradykinin B2-receptor gene (Bk 2r-/-). METHODS AND RESULTS Under basal conditions, tail-cuff blood pressure was higher in Bk2r-/- than in wild-type Bk2r+/+ and heterozygous Bk2r+/- mice (124+/-1 versus 109+/-1 and 111+/-2 mm Hg, respectively; P<.01 for both comparisons), a difference that was confirmed by measurements of intra-arterial blood pressure in unanesthetized mice. Heart weight was greater in Bk2r-/- than in Bk2r+/+ and Bk2r+/- mice (505+/-10 versus 449+/-12 and 477+/-10 mg/100 g body wt, P<.05). Chronic blockade of B2-receptors by Icatibant (50 nmol/100 g body wt twice a day S.C.) or inhibition of nitric oxide synthase by nitro-L-arginine-methyl ester (0.14 mmol/100 g body wt orally) increased the blood pressure of Bk2r+/+ to the levels of Bk2r-/- mice. Compared with the wild-type strain, both Bk2r-/- and Bk2r+/- mice showed exaggerated vasopressor responses to angiotensin II. In addition, chronic administration of an angiotensin AT1-receptor antagonist reduced the basal blood pressure of Bk2r-/- by 21+/-3 mmHg (P<.05) to the levels of Bk2r+/+. No difference was detected between strains as far as plasma renin activity and the expression of renin and AT1-receptor genes are concerned. Chronic salt loading (0.84 mmol/g chow for 15 days) increased the blood pressure of Bk2r-/- and Bk2r+/- by 34+/-3 and 14+/-6 mm Hg, respectively, whereas it was ineffective in Bk2r+/+. CONCLUSIONS Our results suggest that a normally functioning B2-receptor is essential for the maintenance of cardiovascular homeostasis in mice. Dysfunction of the kallikrein-kinin system could contribute to increase blood pressure levels by leaving the activity of vasoconstrictor agents unbalanced.

Cadmium as an Environmental Factor of Hypertension in Animals: New Perspectives on Mechanisms

As already established in human medicine, the relationship between hypertension and environmental factors is becoming increasingly important in veterinary medicine due to the increase in cardiovascular diseases, especially in companion animals (Fox et al., 1999). Therefore, it is very important to study the mechanisms that connect this pathology with the environment as well as the biotic and abiotic factors. Cadmium (Cd) is a ubiquitous environmental pollutant of particular toxicological interest, found above all in industrialized areas. Because of its long biological half-life the toxic effects of cadmium only arise after many years and it concentrates especially in the kidneys, liver, and the central nervous system (Staessen and Lauwerys, 1993). Nowadays, Cd is considered to be an important environmental factor connected with the pathogenesis of arterial hypertension (Glauser et al., 1976; Puri, 1999; Templeton and Cherian, 1983). In order to explain this phenomenon several mechanisms have been proposed including partial agonism with the calcium channels, direct vasoconstrictor action, activation of the sympathetic nervous system, and inhibition of vasodilator substances such as nitric oxide (Cheung, 1984; Demontis et al., 1996). In order to evaluate the possible involvement of other systems in the pathogenesis of hypertension, a strain of genetically selected rats was used because of their low urinary kallikrein excretion. Kallikrein is an enzyme that releases vasoactive peptides (kinins), which in turn produce a decrease in arterial pressure.

Prevention by blockade of angiotensin subtype1-receptors of the development of genetic hypertension but not its heritability.

1 We determined whether early inhibition of angiotensin II subtype1 (AT1) receptors by the newly synthesized nonpeptidic antagonist, A‐81988, can attenuate the development of hypertension in spontaneously hypertensive rats (SHR) and if the altered blood pressure phenotype can be passed on to the subsequent generation, not exposed to the antagonist. 2 Pairs of SHR were mated while drinking tap water or A‐81988 in tap water, and the progeny was maintained on the parental regimen until 14 weeks of age. At this stage, A‐81988‐treated rats showed lower systolic blood pressure and body weight values (136 ±5 versus 185 ±4 mmHg and 247 ±4 versus 283±4 g in controls, P<0.01), while heart rate was similar. In addition, mean blood pressure was reduced (101 ±7 versus 170 ±7 mmHg in controls, P < 0.01), and the pressor responses to intravenous or intracerebroventricular angiotensin II were inhibited by 27 and 59%, respectively. Heart/body weight ratio was smaller in A‐81988‐treated rats (3.2±0.1 versus 3.8±0.1 in controls, P<0.01). 3 The antihypertensive and antihypertrophic effect of A‐81988 persisted in rats removed from therapy for 7 weeks (systolic blood pressure: 173 ±4 versus 220 ±4 mmHg, heart/body weight ratio: 3.4 ±0.1 versus 4.1 ±0.1 in controls at 21 weeks of age, P < 0.01 for both comparisons), whereas the cardiovascular hypertensive phenotype was fully expressed in the subsequent generation that was maintained without treatment. 4 These results indicate that chronic blockade of angiotensin AT1‐receptors attenuates the development of hypertension in SHR but it does not prevent the transmission of hypertension to the following generation. Thus, heritability of the SHRs hypertensive trait is not affected by pharmacological manipulation of the cardiovascular phenotype.

Brain renin-angiotensin system modifies the blood pressure response to intracerebroventricular cadmium in rats.

In order to elucidate the involvement of the brain renin-angiotensin system (RAS) in cadmium intracerebroventricular (ICV) hypertension, we evaluated the effects of a pretreatment with different drugs: clonidine, an α2 adrenergic agonist, enalapril and captopril, both ACE inhibitors, and saralasin, a competitive nonselective AT1 and AT2 receptor antagonist. We used a rat strain with low levels of kallikrein (LKR) that was more sensitive to ICV cadmium hypertension, compared with normal kallikrein rats (NKRs), the control strain. The interplay between the kallikrein-kinin system and the RAS in the LKR strain caused various hemodynamic alterations, which we believe were the result of elevated RAS activity in these animals. Moreover, we suggest that the defective kallikrein-kinin system in LKR may also cause an alteration in the activation of brain RAS in these animals. The LKR displayed elevated concentrations of plasma AII, hypertrophy of the myocardium, and initial alterations in the renal glomerulotubular system. With the exception of clonidine, all of the other drugs showed greater antihypertensive effects of differing statistical significance in LKR, compared with NKR. Both ACE inhibitors were able to significantly reduce pressor response to cadmium ICV in LKR throughout the experiment, whereas in NKR, they were only able to reduce the hypertensive peak of cadmium. A significant protective effect was also observed in LKR pretreated with saralasin, while no effect was observed in NKR. These findings confirm the presence of brain RAS activation in LKR and its contribution to the central control of pressor response to cadmium ICV.

Different response of intracerebroventricular cadmium administration on blood pressure in normal and low urinary kallikrein rats

Cadmium intracerebroventricular (i.c.v.) administration, at definite concentrations, induces a dose-dependent increase in the systemic blood pressure. Kallikreins are suggested to be important regulators of cardiovascular function. We evaluated the effects of 10 microg i.c.v. cadmium on mean blood pressure (MBP) and several urinary parameters such as 24h urine volume, sodium and potassium excretion and osmolality in a rat strain inbred for low urinary kallikrein and in normal-kallikrein Wistar rats. Low-kallikrein rats (LKR) showed an increase in MBP that, after an initial peak (27% from baseline), persisted higher than basal levels (10%) over 24h. In normal-kallikrein rats (NKR) a different reaction of blood pressure to cadmium was observed, causing a temporary increase (26% from baseline) of the systemic blood pressure, that returned to normal values within 2h. In addition, LKR showed a considerable reduction in the urinary volume (UV; 43.0+/-20 ml/24h versus 13.2+/-6 ml/24h, P<0.006), with an increase in the urinary osmolality (U(Osm); 500+/-210 mOsm/l versus 1391+/-245 mOsm/l, P<0.0002). Sodium (U(Na); 1761+/-432 microEq/24h versus 1156+/-522 microEq/24h, P<0.03) and potassium excretion (U(K); 2186+/-482 microEq/24h versus 936+/-299 microEq/24h, P<0.0006) were both significantly reduced. No changes in UV, U(Osm) and U(K) were observed in normal urinary kallikrein rats with the exception of U(Na) excretion that was significantly increased (667+/-274 microEq/24h versus 1725+/-300 microEq/24h, P<0.03). These results suggest that a genetically determined defect in urinary kallikrein excretion leads to a different hypertensive response to i.c.v. cadmium and to a different renal excretion of electrolytes. Perhaps the differences of blood pressure response could be due, at least in part, to a different sensitivity of LKR to cadmium: this implies a complex and articulate hypertensive effect of cadmium involving more systems than those supposed so far.

Quantitative assay for bradykinin in rat plasma by liquid chromatography coupled to tandem mass spectrometry

An assay to quantify bradykinin in rat plasma has been developed and validated, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sar-D-Phe(8)-des-Arg(9)-bradykinin was used as internal standard. Aprotinin was added to rat plasma to inhibit the activity of proteinases. Recoveries for solid-phase extraction (SPE) on Strata X reversed phase were greater than 80%. Multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer equipped with an electrospray source (ESI), operating in the positive ion-mode, was used for detection. The assay was validated and stability was explored. Bradykinin (10-500 ng/mL) was quantified with accuracy values (% RE) below 10% and intra- and inter-day precisions (% RSD) below 12 and 16%, respectively, for all concentrations. The method was successfully applied to several plasma samples from low levels kallikrein rats (LKRs) compared with normal kallikrein rats (NKRs).

Acute Effects of Furosemide on Blood Pressure in Functionally Anephric, Volume-Expanded Rats

The hypotensive effect of loop diuretics is primarily mediated by a decrease in extracellular fluid volume that follows the intense diuresis. In the present study we investigated whether furosemide (1.25 mg/kg i.v.) can acutely lower blood pressure independently of its diuretic action. The experiments were performed in four groups of male Wistar rats: (a) rats in which furosemide was administered after bilateral ligation of renal vessels and saline (0.9% NaCl at a rate of 0.25 ml/min to a final volume of 1% of body weight) infusion; (b) rats in which furosemide was injected after bilateral ligation of ureters and saline infusion; (c) rats pretreated with indomethacin (3 mg/kg i.p. for 4 days) in which furosemide was injected after bilateral ligation of ureters and saline infusion; (d) sham-operated rats with intact kidneys. All the groups were compared with appropriate time controls. After furosemide injection, heart rate and blood pressure were serially recorded for 30 min by plethysmography. Hematocrit, plasma 6-keto-prostaglandin F1 alpha levels and plasma renin activity were measured at baseline and 20 min after furosemide injection. No change in blood pressure followed furosemide administration in rats with ligation of the renal vessels. Conversely, furosemide induced a rapid and significant fall in blood pressure associated with a significant increase in heart rate in rats with ligation of the ureters and rats with intact kidneys. Indomethacin pretreatment prevented the furosemide-induced decrease in blood pressure in rats with ligation of the ureters. Hematocrit and plasma 6-keto-prostaglandin F1 alpha levels were not affected by furosemide in either group.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracranial volume receptors: possible role on ADH homeostatic control

Volume receptors are situated in many organs and are capable of modulating ADH secretion. We have evaluated the variation of plasma ADH concentration after an experimentally induced increase of cerebrospinal fluid (CSF) pressure (PCSF). The experiment was performed in controlled environmental conditions to avoid pain or stress-related ADH release. In 15 rats (10 experimental, 5 control) a cannula was positioned in the left cerebral ventricle: in the experimental group artificial CSF was infused at a rate of 0.6 (µl/min for 6h: this manoeuvre, in a separate set of animals obtained an increase from 13.03±0.8 to 25.4±2.5 cmH2O of Pcsf. The same conditions were reproduced in the control group without infusion into lateral ventricle. At the end of the experiment, plasma ADH had fallen significantly in the experimental group from 18.9±4.8 to 11.9±2.3 pg/ml (p<0.05), while it was not changed in the control group (from 25.5±13.7 to 23.7±16.2 pg/ml). Heart rate, arterial pressure, plasma Na+ and osmolality, did not change significantly. Plasma K+ fell significantly in both groups: from 5.5±0.6 to 4.3±0.3 (p<0.05) and from 5.4±0.7 to 4.3±0.15 mEq/l (p<0.05) in the experimental and control group respectively. Plasma creatinine was normal, checked only at the end of the experiment. Our results demonstrate that a relationship exists between PCSF variations and plasma ADH concentration. We believe this relationship is due to the pressure receptors in the cerebral ventricles or in structures connected to it, such as the inner ear, and we hypothesize the existence of a control system of body fluids, more diffused than thought to be, up till now.

Investigation of the effects of Lycium barbarum polysaccharides against cadmium induced damage in testis

This study describes the effects of Lycium barbarum polysaccharides (LBP) on testicular damage induced by cadmium (Cd). Adult male rats were i.p. injected with CdCl2 (4mg/Kg, once) with or without LBP pretreatment (300mg/Kg orally, once a day, for 30days). Testis weight, morphological/histological structure and oxidative stress parameters were evaluated. Several adverse effects were observed after CdCl2 injection, with a significant decrease in body/testis weight ratio (P<0.05), gross morphological changes with hyperemia of the parenchyma, increased volume and alteration in the structure of the seminiferous tubules. Furthermore, Cd intoxication caused a significant decrease of glutathione (GSH) and Trolox equivalent antioxidant capacity (TEAC) in testis (P<0.05) together with a significant increase (P<0.01) of 3-nitro-l-tyrosine (3NT) while malondialdehyde (MDA) did not change. LBP pretreatment caused slight signs of improvement in the morphology of the seminiferous tubules. Our results confirm that Cd induces testicular damage and suggest the oxidative stress involvement. LBP could ameliorate Cd testicular damage but further investigations are needed.

Role of B1 kinin receptors in pain response in low kallikrein wistar rats

Kinins are potent autacoids which are primarily involved in inflammatory and pain processes (Calixto et al., 2000). They are generated by enzymatic cleavage of kallikrein and exert their biological effects locally, through the activation of two receptors named B1 and B2. Recent data suggest that they act not only locally but also as cerebral mediators in the control of nociception. However, at the moment neither the role nor the topographical distribution of kinin receptors is clear, either with regard to nociception or with regard to other functions. Many authors have suggested that B2 receptors are constitutively expressed while B1 are generally absent in normal tissues and are induced following tissue injury and/or by the action of inflammatory agents. The B2 receptors are prevalent in the acute phase of pain response and B1 in the chronic phase (Hall and Morton, 1997). However, recent research has indicated hypoalgesia in B1–receptor knockout mice against acute nociception induced by heat stimuli (Rupniak et al., 1997). At the moment the role played by these receptors is not clear because it depends in part on the method used to study pain response. A useful way to study pain response may be by experimenting with a rat strain with low renal excretion of kallikrein (Madeddu et al., 1996). The chronic presence of low levels of renal kallikrein in such rats may have modified the expression of the kinin receptors, and thus also their pain response, with respect to normal rats. The aim of this work was to quantify the expression of B1 receptors in this strain of rats in response to heat stimuli, both in terms of their basal pain response and after pre-treating them with a B1 receptor antagonist. The hot plate test was used to do this.