Effect of dopamine antagonists on the urine flow of rats infused with hypotonic saline.

1. The probable involvement of dopamine in the regulation of water excretion was investigated by administering dopamine antagonists intravenously to barbiturate--anaesthetized rats undergoing a water diuresis induced by the infusion of 0.83% glucose with 0.3% NaCl at the rate of 9 ml h-1. 2. Administration of 100 micrograms of the D1-/D2-dopamine antagonist, haloperidol, reduced the enhanced urine flow of rats infused with the hypotonic solution by 69% (from 75.4 +/- 13.0 to 23.6 +/- 6.0 microliter min-1, P less than 0.01). Similarly, the D1-receptor antagonist, SCH 23390, reduced urine flow by 58% (from 77.5 +/- 9.2 to 32.7 +/- 7.2 microliters min-1, P less than 0.01) and the D2-receptor antagonist, sulpiride, by 47% (from 66.2 +/- 8.6 to 35.1 +/- 6.8 microliter min-1, P less than 0.05). 3. The injection of SCH 23390 increased the urine osmolality from 189.6 +/- 27.5 to 479.8 +/- 45.8 mosm kg-1 (P less than 0.05). There was no significant change in sodium and potassium excretion in any of the experiments. Blood pressure (BP) decreased after haloperidol and SCH 23390 injection from control values of 121.7 +/- 1.7 and 116.5 +/- 7.4 to 113.3 +/- 3.3 and 106.0 +/- 8.8 mmHg respectively (P less than 0.05). 4. To study whether the influence of dopamine antagonists on urine flow during water diuresis depends on antidiuretic hormone (ADH), we administered 0.6 micrograms d(CH2)5-D-Phe-Ile-AVP (an ADH antagonist) shortly after the injection of 100 micrograms SCH 23390. The preferential V2 ADH-antagonist abolished the antidiuretic effect of SCH 23390 but did not affect its blood pressure reducing effect (from 118.6 +/- 5.6 to 103.2 +/- 4.6 mmHg, P <0.01). 5. These results suggest that dopamine antagonists blunted the hypotonic saline-induced diuresis by favouring ADH release through an interference with an inhibitory dopaminergic pathway.

The influence of tetracosactide and adrenal steroids on renal kallikrein activity and urinary kallikrein excretion in rats.

The effect of adrenal steroids (mineralo- and glucocorticoids) as well as that of the adrenocorticotrophic peptide tetracosactide (beta 1-24 corticotropin) on the renal kallikrein activity and on the urinary kallikrein excretion of rats was investigated. After the animals had been adapted to metabolic cages, they were injected with deoxycorticosterone acetate (15 mg/kg day), corticosterone (40 mg/kg day), both steroids combined or the vehicle (sesame oil). Additional groups of rats received tetracosactide (0.05, 0.1 or 0.2 mg/day) or the vehicle (100 microliter of 38 X 10(-3) M ZnCl2). After four days of treatment the urinary kallikrein excretion was higher in deoxycorticosterone-treated rats than in their controls. This increase was prevented when corticosterone was administered simultaneously. The renal kallikrein activity of corticosterone as well as that of deoxycorticosterone plus corticosterone-treated rats was subnormal. A dose-related reduction of both the renal kallikrein activity and the urinary kallikrein excretion was observed 2 days after starting the tetracosactide administration. It may be concluded that a stimulation of the endogenous release of glucocorticoids in the rat reduces the renal kallikrein activity and that glucocorticoids can prevent the stimulating effect of mineralocorticoids.

The influence of isotonic saline administration on the urinary excretion of kallikrein in rats.

Acute saline loading is known to increase kallikrein excretion. To clarify whether this is a specific stimulatory effect or rather a non-specific wash-out, pentobarbital anesthetized rats were loaded thrice (5 min infusions) at 40 min intervals with a volume of 150 mM NaCl equal to 5% of their body wt. The effect of such a load on the central venous pressure was studied in a separate group of rats. Control animals did not receive the infusions. Kallikrein excretion (amidolytic assay) increased with the first, but decreased with the subsequent saline administrations. The rise observed after the first load lost significance when kallikrein excretion was related to that of creatinine. The reduction observed after the second and third infusions remained significant even when expressed per mg of creatinine. Thus, saline load induced kallikrein "stimulation" is due to a non-specific wash-out. Similar transient enhancements of central venous pressure were observed after each of the three loads. This, together with the unchanged creatinine excretion (except for the rise seen after the first load) indicate that the lack of kallikrein stimulation after the second and third loads was not due to the appearance of heart failure. Saline loaded rats had a renal kallikrein activity at the end of the experiment which did not differ from that of controls. Plasma aldosterone concentration was reduced in saline infused rats, and it correlated with the kallikrein excretion when both, NaCl loaded and control rats, were taken into account.

Video-imaging microfluorometry identifies alpha- and beta-like cell types in Madin-Darby canine kidney monolayers.

On the basis of intracellular, accumulation of c-SNAFL-2, we have identified three cell subtypes in Madin-Darby canine kidney (MDCK) monolayers. Highly fluorescent cells (HFC) have a high intracellular pH (pHi, whereas cells with medium fluorescence (MFC) have low pHi when perfused with buffer containing 125 mM Cl-. HFC express a Cl-/HCO3- exchanger on the apical but not the basolateral membrane. MFC express a Cl-/HCO3- exchanger on the basolateral but not the apical membrane. We have termed these cells beta- and alpha-MDCK cells, respectively. Cells with low fluorescence (LFC) probably extrude c-SNAFL-2 through a monocarboxylate transporter, because p-4-(chloromercuri)phenylsulfonic acid (PCMBS), an inhibitor of this transporter, leads to homogeneous fluorescence.

Influence of extracellular pH on intracellular pH in arterioles and skeletal muscle of spontaneously hypertensive and Wistar-Kyoto rats.

The effect of changing extracellular pH (pHo) on intracellular pH (pHi) in mesenteric arterioles of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats was investigated using the fluorescent indicator seminaphthorhodafluor c-SNARF-1 to measure pHi. Although the pHi of arterioles from WKY varied directly with pHo, that of SHR arterioles varied inversely. This abnormal pHi regulation in SHR was corrected by 50-microM 5-(N-ethylisopropyl)amiloride (EIPA). Vmax of H+ transport in response to changing extracellular Na+ was higher in SHR than in WKY, but Km did not differ. The Hill coefficient for H+ transport with respect to pH; was 1.69 in prehypertensive and 1.56 in hypertensive SHR. These results indicate that the Na+/H+ exchanger is particularly hyperactive in mesenteric arterioles of SHR.

Reverse transcriptase-polymerase chain reaction study of anion exchanger-2 in canine tissues and different Madin-Darby canine kidney cell types.

Reverse transcriptase-polymerase chain reaction (RT-PCR) of mRNA from canine large intestine, skeletal muscle, pancreas, kidney, and spleen and from cultured wild-type and C7 and C11 Madin-Darby canine kidney (MDCK) cells revealed considerable variation in anion exchanger (AE)1 and AE2 mRNA levels between the tissues. Similar high levels of AE2 mRNA were detected in all the MDCK cell populations. AE2 in MDCK cells is probably the basolateral Cl-/HCO3- exchanger common to the principal and beta-intercalated cells.

Effect of dihydralazine on the renal kallikrein-kinin system of the rat.

Dihydralazine (0.1 mg/kg), injected intravenously into male Sprague-Dawley rats, caused a decrease in mean arterial blood pressure and an increase in renal plasma flow, while urine volume remained unchanged. Dihydralazine had no effect on kallikrein excretion in the urine and on kallikrein activity in the renal cortex. No correlation was found between renal kallikrein and either renal plasma flow or mean arterial blood pressure. The excretion of kinins in the urine rose markedly after the administration of dihydralazine; no correlation between urinary kinins and urinary or renal kallikrein was observed. Dihydralazine had no influence on the kininogen content of blood-free renal cortex. The enzymatic activity of kininase II in renal cortex was not impaired by dihydralazine. It is suggested that the increased formation of kinins within the kidney could be involved in the vasodilating and blood pressure lowering effect of dihydralazine.

SA446, a new orally active converting enzyme inhibitor: antihypertensive action and comparison with captopril in stroke-prone spontaneously hypersensitive rats.

The converting enzyme inhibitors (CEI) SA446 and Captopril (CAP) were given orally at a dose of 50 mg/kg per day to adult stroke-prone spontaneously hypertensive rats (SHRSP) over a period of four weeks. Both CEI lowered arterial blood pressure (BP) to a similar extent. CAP was more inhibitory on the plasma renin-angiotensin system (RAS) than SA446. Both CEI lowered urinary aldosterone excretion but had little (SA446) or no (CAP) natriuretic effect. CAP reduced the pressor responses to intravenous (i.v.) angiotensin I (ANG I) more (52%) than SA446 (18%) and potentiated the depressor responses to i.v. bradykinin more (fortyfold) than SA446 (tenfold). In contrast, SA446 treatment reduced the pressor responses to intracerebroventricular (i.c.v.) ANG I by 21% and led to a rise in the hypothalamic renin concentration. Oral CAP treatment for four weeks did not produce these signs of a brain converting enzyme inhibition. It is concluded that SA446 is equally as antihypertensive as CAP in SHRSP. SA446 appears to penetrate more readily into the brain and to exert its action partly through inhibition of the brain RAS which is known to be stimulated in SHRSP.

Reduced glutathione inhibits rabbit and rat skeletal muscle lactate dehydrogenase and prevents dinitrophenol induced extracellular acidification by an epithelial cell line.

Glutathione (GSH) is a tripeptide synthesised enzymatically from its components amino-acids by unicellular and multicellular organisms. GSH acts as a cellular anti-oxidant, protects the structural configuration of some enzymes, is involved in erythrocyte function and plays a role as co-enzyme in several reactions. We have found that GSH inhibits purified lactate dehydrogenase (1.56 U LDH/ml) from rabbit skeletal muscle after 6 min pre-incubation with an ED50 of about 5.4 microM. The inhibition is time dependent with a maximum after 45 minutes pre-incubation. Buffer (5 x 10(-2) M TRIZMA hydrochloride, pH 7.4) and a chelator (2 x 10(-3) M EDTA) in the pre-incubation solution did not prevent the inhibition. Prolonged dialysis was almost without effect on GSH-inhibited LDH activity solution, indicating either an irreversible or a very tight binding inhibition. Kinetic analysis showed that this inhibition is of a very tight binding and at the same time of the uncompetitive type. GSH also inhibits LDH activity of rat M. soleus and M. gastrocnemius homogenates. This effect is probably unrelated to the reducing property of GSH since dithioerythritol (0.17-1.34 mM) does not mimic it. Loading of MDCK cells with glutathione ethylester completely prevented the acidification induced by 2,4-dinitrophenol, suggesting that GSH may influence the glycolytic pathway in vivo.

Dopamine receptor antagonists inhibit the natriuretic response to atrial natriuretic factor (ANF).

The diuretic and natriuretic response of anesthetized rats to low doses of semi-purified atrial extracts or synthetic alpha-hANP was completely blocked by intravenous injection of 50 micrograms of haloperidol or chlorpromazine. Sulpiride or metoclopramide at the same doses did not show this effect. We conclude from these results that dopamine receptors, probably of the D1-type, are involved in the natriuretic effect of the atrial peptides.

Imaging positron emitting radionuclides generated during radiation therapy.

In vivo generated positron emitting radioisotopes, primarily C-11 and N-13, have been documented following therapy with accelerators larger than 10 MeV. Six patients had positron emission tomography 15 to 25 minutes after radiation therapy with a 42 MeV accelerator. Five patients had recurrent colorectal malignancy, and one required therapy for a carcinoma of the common bile duct. We sought to determine whether state-of-the-art PET technology could be used to monitor the three-dimensional activity distribution of radiation-induced radioactivity. At the time of the examination all six patients had sufficient concentrations of C-11 and N-13 activity in the irradiated volume to permit the evaluation of the activity distribution. We found significant activity at the body surface, which permitted field delineation. We conclude that the in vivo generated radioactivity can be monitored with PET.

Interrelation between renal kallikrein and diuresis in rats.

In rats diuresis was produced by 1. furosemide (5 mg/kg/s.c.), 2. triamterene (50 mg/kg/p.o.), 3. mannitol (250 microliters, 30%) and 4. change (+ 35 mm Hg) in perfusion pressure in the isolated perfused rat kidney. In all experiments a prolonged increase in diuresis and natriuresis was effected. Urinary potassium excretion was markedly enhanced by furosemide and change in perfusion pressure, moderately increased by mannitol, and significantly depressed by triamterene. Urinary kallikrein excretion, however, showed in all of the experiments a biphasic course with an initial increase and secondary decrease. The increase of kallikrein excretion was observed only in the periods when urine flow started to increase. Thus, no correlation was found between kallikrein excretion and either urine volume or urinary sodium excretion. Kallikrein excretion correlated with potassium excretion in urine after furosemide, triamterene, and mannitol, however, it did not after increase in perfusion pressure. In renal cortical tissue kallikrein activity was reduced after all experiments, independently of the mechanisms, diuresis was induced.

Role of perfusate hydrogen ion activity in kallikrein release from isolated rat kidneys.

The present experiments were performed to investigate whether renal kallikrein release by isolated perfused rat kidneys correlates with acid-base-related parameters. Kallikrein excretion per millilitre of glomerular filtrate was inversely correlated with perfusate pH (r = -0.49, P < 0.001) and HCO3- concentration (r = -0.46, P < 0.005). A direct relationship between kallikrein excretion per millilitre of glomerular filtrate and urinary Na+/K+ ratio was found (r = 0.59, P < 0.001). Some 86% of the variability (F ratio 110, P < 0.00001) of urinary kallikrein activity was attributable to the perfusate pH and the urinary cation ratio. Therefore, urinary kallikrein activity was highly correlated with perfusate H+ activity corrected by the urinary Na+/K+ ratio (r = 0.92, P < 0.0001). Kallikrein secretion into the distal tubular fluid appears to be regulated by blood H+ activity, and modulated by factors that affect the distal Na+ and K+ handling. The HCO3 - excretion rate was inversely correlated with the urinary kallikrein activity (r = -0.62, P < 0.001). This finding confirms previous data from the author's laboratory showing a kallikrein involvement in the regulation of HCO3- secretion in rats and rabbits. Kallikrein probably transduces the sensing of interstitial fluid H+ activity by the connecting tubule cells into an appropriate translocation of HCO3- or H+ to the tubular lumen by the intercalated cells.

Measurement of kallikrein activity in urine of rats and man using a chromogenic tripeptide substrate. Validation of the amidolytic assay by means of bradykinin radioimmunoassay.

Urinary kallikrein activity in rats and man was measured by an amidolytic assay using the chromogenic tripeptide D-valyl-leucyl-arginine-p-nitroanilide (S 2266). The sensitivity of the assay permits the measurement of 3.5 to 250 mKU of purified porcine pancreatic kallikrein. The intraassay coefficient of variation was 1.6 +/- 0.3% and the interassay coefficient of variation 7.4 +/- 0.9%. No changes in kallikrein activity was found in urine samples kept for 7 days at 4 degrees C for 14 months at -20 degrees C. Repeated freezing and thawing reduced kallikrein activity. Kallikrein activity of the same urine samples was also estimated by means of a bradykinin radioimmunoassay, which determines the kinins released during incubation of urine with partially purified dog kininogen (kininogenase activity). The urinary kallikrein activity assayed by means of these two methods showed a high correlation with coefficients of r = 0.9134 for rat urine (p less than 0.0001) and r = 0.8002 for human urine (p less than 0.001).

Urinary kallikrein response to repeated frusemide injections in rats: effect of adrenalectomy and deoxycorticosterone acetate treatment.

The effect of repeated intravenous injections of frusemide (0.17 mg/kg) on the urinary kallikrein excretion of normal, adrenalectomized and deoxycorticosterone acetate (DOCA)-treated adrenalectomized rats was studied. Adrenalectomy decreased baseline urinary kallikrein excretion, while DOCA treatment restored it to normal. Frusemide injections repeatedly increased urinary kallikrein excretion in the three groups of rats studied. Compared with control and DOCA-treated animals, adrenalectomized rats were less responsive. The excretion of urinary kallikrein was positively correlated with urine volume and with the excretion of sodium and potassium in all groups. The regression lines were shifted to the left in DOCA-treated adrenalectomized rats showing that mineralocorticoids enhance the effect of frusemide on urinary kallikrein excretion. The regression lines between urinary kallikrein excretion and the measured variables in adrenalectomized rats did not differ from those of control animals. We conclude that the response of urinary kallikrein to frusemide is influenced by the level of mineralocorticoid activity. The effect of frusemide on urinary kallikrein excretion does not appear to be a 'wash-out' of the enzyme since its influence did not subside after repeated injections.