Philip J. Privitera

Bartter's Syndrome: Urinary Prostaglandin E-like Material and Kallikrein; Indomethacin Effects

The urinary excretions of prostaglandin E-like material (iPGE) and kallikrein were measured in two children with Bartters syndrome. Urinary iPGE excretion was three and 10 times greater than normal, and urinary kallikrein was five and 10 times greater than normal in the two subjects. Furthermore, excretions of iPGE and kallikrein were highly correlated (P less than 0.005) with each other before and during treatment with indomethacin, a prostaglandin synthetase inhibitor. Indomethacin significantly (P less than 0.001) reduced urinary iPGE, urinary kallikrein, and plasma renin activity, while increasing the sensitivity to intravenous angiotensin II and the serum potassium to normal. The results confirm that renal prostaglandins may be involved in the pathogenesis of Bartters syndrome and suggest that renal prostaglandins and the kallikrein-kinin system are linked.

Childhood familial and racial differences in physiologic and biochemical factors related to hypertension.

AR Hohn, DA Riopel, JE Keil, CB Loadholt, HS Margolius, PV Halushka, PJ Privitera, related to hypertension Childhood familial and racial differences in physiologic and biochemical factors ISSN: 1524-4563 Copyright

Rostral ventrolateral medulla as a site for the central hypertensive action of kinins.

In the present study, we focused on the rostral ventrolateral medulla as a possible site of action for kinins because of its established importance in the central regulation of the cardiovascular system. Unilateral microinjections of 100 pmol to 4 nmol bradykinin into the rostral ventrolateral medulla produced dose-dependent increases in mean arterial pressure in Sprague-Dawley (SD) rats, Wistar-Kyoto (WKY) rats, and spontaneously hypertensive rats (SHR). The dose-response curves for the hypertensive responses to bradykinin in SD and WKY rats were essentially the same, whereas the hypertensive effect of bradykinin was significantly greater in SHR than in either SD or WKY rats. The kinin B2 receptor antagonists D-Arg0,Hyp3,Thi5,8,D-Phe7-bradykinin and Hoe 140 inhibited the hypertensive responses to bradykinin in both SHR and WKY rats. The hypertensive effect of 500 pmol bradykinin was reduced 65 +/- 5% after 4 nmol of D-Arg0, Hyp3,Thi5,8,D-Phe7-bradykinin in SHR and 50 +/- 16% in WKY rats, whereas 1 nmol Hoe 140 abolished the hypertensive effect of 500 pmol bradykinin injected into the rostral ventrolateral medulla. Microinjection of D-Arg0,Hyp3,Thi5,8,D-Phe7-bradykinin produced prolonged dose-dependent decreases in mean arterial pressure and heart rate. Blood pressure decreased 70 +/- 8 mm Hg and heart rate decreased 49 +/- 9 beats per minute in SHR, whereas in WKY rats mean arterial pressure decreased 12 +/- 4 mm Hg, with no change in heart rate. In a similar fashion, Hoe 140 caused a 51 +/- 7 and 17 +/- 3 mm Hg reduction in blood pressure in SHR and WKY rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative determination of propranolol, propranolol glycol and N-desisopropylpropranolol in brain tissue by electron capture gas chromatography.

A method for the quantitative determination of propranolol and two of its active metabolites, 3-(alpha-naphthoxy)-1,2-propanediol (propranolol glycol) and N-des-isopropylpropranolol, in brain tissue of mice is described. Tissues are homogenized in perchloric acid-acetonitrile. Propranolol and its metabolites are isolated from the supernatant by solvent extraction and separated and detected as their trifluoroacetyl derivatives by electron capture gas chromatography. Chemical structures of the derivatives were confirmed by gas chromatography-mass spectrometry. The electron capture detector response of all three compounds is high, 0.7-2.0 X 10(-16) moles/sec. Brain levels of 10-250 ng/g can be detected of all three compounds with high specificity and good precision.

Effects of centrally administered propranlol on plasma renin activity, plasma norepinephrine and arterial pressure

Abstract Single doses of d,l-propranolol (0.1–0.5 mg/kg) administered intarcisternally (IC) to anesthetized dogs produced dose-dependent decreases in plasma renin activity and mean arterial pressure whereas identical doses given intravenously had no significant effect on these parameters. Intracisternal d,l-propranolol also significantly reduced circulating norepinephrine levels concomitant with the renin suppressing and hypotensive effects. Acute renal denervation abolished the renin suppressing, but not the hypotensive effect of intracisternal d,l-propranolol. Both d- and l-propranolol administered intracisternally significantly lowered plasma renin activity and arterial pressure to a similar degree, but intracisternal lidocaine had no effect on these parameters. These results indicate that propranolol can suppress plasma renin activity through an action within the central nervous system and suggest that both the renin suppression and hypotension produced by intracisternal propranolol resulted from a centrally mediated reduction in peripheral sympathetic activity. The equal potency of the stereoisomers of propranolol and the observed differences between the effect of propranolol and lidocaine suggest that these central actions involve a mechanism that may be independent of beta receptor blockade or the local anesthetic properties of drug.

Brain Kallikrein-Kinin System Abnormalities in Spontaneously Hypertensive Rats

The objective of the present study was to determine whether the brain kallikrein-kinin system differs between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) and if so, whether any detected differences occur before the development of hypertension in SHR. We measured cerebrospinal fluid levels of various components of the system in adult and young prehypertensive SHR and WKY. Cerebrospinal fluid kinin concentration and appearance rate were higher in SHR. Cerebrospinal fluid active kallikrein level and kininogenase activity were also higher in adult SHR. In addition, cerebrospinal fluid kinin concentration and appearance rate were higher in prehypertensive, 5- to 6-week-old SHR compared with age-matched WKY. However, no differences in cerebrospinal fluid kallikrein or kininogenase activity were observed between the two strains of young rats. Cerebrospinal fluid kinin concentration was higher in young versus adult rats of the same strain. In WKY, cerebrospinal fluid kallikrein also decreased with age although cerebrospinal fluid kallikrein concentration did not decrease in young and adult SHR. Together, these data suggest that there is a hyperactive kallikrein-kinin system in the brain of SHR that may contribute to the hypertensive state in this animal model.

Cerebrospinal fluid kallikrein in spontaneously hypertensive and desoxycorticosterone acetate-salt hypertensive rats.

Objective: To determine whether immunoreactive tissue kallikrein levels in cerebrospinal fluid (CSF) of spontaneously hypertensive rats (SHR) and desoxycorticosterone acetate (DOCA)—salt-treated hypertensive rats are elevated compared with normotensive Wistar—Kyoto (WKY) and Sprague—Dawley rats. Design: The present study was designed to test the hypothesis that the activity of the brain tissue kallikrein—kinin system is enhanced in hypertensive states. Methods: Age-matched 18- to 19-week-old SHR and WKY rats, and Sprague—Dawley rats treated for 6 weeks either with 2 mg/kg per day DOCA subcutaneously and 0.9% saline in the drinking water, or with vehicle and tap water to drink, were studied. CSF was collected from a cannula inserted into the cisterna magna, and was frozen until the tissue kallikrein in the samples was measured by radioimmunoassay. Arterial pressure in the SHR and WKY rats was measured directly via a cannula inserted in the femoral artery or by tail-cuff plethysmography. Results: In adult 18- to 19-week-old SHR the CSF kallikrein concentration was higher than in WKY rats. The CSF flow rate in SHR was also higher than in WKY rats. The rate of appearance of kallikrein in the CSF of SHR was twice that in WKY rats. Moreover, CSF kininogenase activity in SHR was significantly higher than that in age-matched WKY rats. In DOCA—salt hypertensive rats the CSF kallikrein concentration was higher than in vehicle-treated control rats. Acute elevation of blood pressure with a 120-min intravenous phenylephrine infusion did not change the CSF kallikrein concentration in 50 rats compared with vehicle-treated control rats. This is the first study to quantitate immunoreactive tissue kallikrein in the CSF of rats and to show elevated levels of CSF kallikrein in hypertensive rats compared with normotensive rats. Conclusion: The present data suggest that higher brain kallikrein activity in hypertensive rats may play a role in the development of elevated blood pressure.

Autoradiographic localization of [125I-Tyr0]bradykinin binding sites in brains of Wistar-Kyoto and spontaneously hypertensive rats.

Abstract1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).2. Serial sections of brains were cut from adult WKY and SHR and specific [125I-Tyr0]bradykinin ([125I-Tyr0]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques.3. Specific binding of [125I Tyr0]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85–90% of total binding) was of high affinity and saturable with KD values in the range of 100 pM and a Bmax of 0.75 fmol per mg tissue equivalent in the NTS–X–AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg9-BK. The B2 receptor antagonist icatibant inhibited [125I-Tyr0]BK binding with a Ki value of 0.63 ± 0.19 nM in WKY and 0.91 ± 0.73 nM in SHR, while Ki values for the B> 1 receptor agonist DesArg9-BK were 1475 ± 1055 and 806 ± 362 nM in WKY and SHR, respectively.4. Our finding of specific high-affinity [125I-Tyr0]BK B2 binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B2 receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected.

Utilization of a radioimmunoassay to detect the generation of Arg-Pro-Pro-Gly-Phe, a stable endproduct of bradykinin metabolism (from cultured rat aortic smooth muscle cells exposed to bradykinin).

The peptide hormone bradykinin is a mediator in many physiological and pathological processes. The generation and, to a limited extent, metabolism occur at the sites of action. The short half-life of bradykinin (approximately 15 s) renders measurements of its concentration in bodily fluids difficult. As a result, recent methods utilizing either ELISA or HPLC/mass spectrometry, have been developed for the measurement of the stable metabolic endproduct of bradykinin, i.e., the pentapeptide Arg-Pro-Pro-Gly-Phe (RPPGF; BK[1-5]). Both have been successfully used to quantitate levels of RPPGF in plasma, pulmonary and nasal exudates. In this study, we demonstrate the characterization and utility of a radioimmunoassay for the measurement of RPPGF, using a newly synthesized radioiodinated analogue of RPPGF, i.e., RPPG(125I)F. This radioimmunoassay shows an IC50 of 80.5 +/- 7.4 pg/tube (n = 23) with a linear range of detection between 10 and 500 pg/tube. The assay was used to demonstrate the time-dependent generation of RPPGF by cultured rat aortic vascular smooth muscle cells exposed to bradykinin (100 ng/ml). Peak generation of RPPGF was 74.5 +/- 9.7 pmol/well (n = 5) after 24 h of incubation. Captopril, an inhibitor of angiotensin-converting enzyme (kininase II), inhibited generation in a concentration-dependent manner. The results characterize a new radioimmunoassay for the stable metabolic endproduct of bradykinin, RPPGF.

Hypertensive effect of tissue kallikrein in rostral ventrolateral medulla is mediated by brain kinins

Microinjections of kallikrein, 0.5-2.0 units, in the rostral ventrolateral medulla (RVLM) of brain increased arterial pressure in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). This effect was significantly greater in SHR. The kinin B2 receptor antagonist icatibant (Hoe 140) blocked the hypertensive responses to kallikrein in both groups and caused greater hypotension and bradycardia in SHR. These results suggest that local kinins in the RVLM act to alter cardiovascular function and may be involved in the maintenance of blood pressure in the SHR.