Mechanisms of the inhibition of human erythrocyte pyridoxal kinase by drugs.

The aim of this study was to investigate the interaction between drugs chosen for their clinical neurotoxicity or chemical structure and vitamin B6 metabolism. After a preliminary screening of drugs to determine their potential inhibitory effect on erythrocyte nonpurified pyridoxal kinase (PLK) (EC 2.7.1.35), additional investigations, including kinetic studies and detection of chemical reactivity between the inhibiting drugs and pyridoxal (PL) or pyridoxal-5'-phosphate (PLP), using UV-visible spectrophotometry and mass analysis, were carried out to specify the mechanism of PLK inhibition. Depending on the results, the inhibiting drugs were divided into three groups. The first group included theophylline and progabide and inhibited PLK using either PL or pyridoxamine (PM) as substrate and thereby were true inhibitors. Moreover, they did not form covalent complexes with PL or PLP. The second group, which included cycloserine, dopamine, isoniazid, and thiamphenicol glycinate, inhibited PLK using PL, but not PM, as substrate. They were able to react with PL or PLP to form covalent complexes, and kinetic studies suggested that the observed PLK inhibition was due to these formed complexes. A third group, which consisted of levodopa, D-penicillamine, and muzolimine, inhibited PLK using PL, but not PM, as substrate. They formed, with PL or PLP, chemical derivatives that probably had no inhibitory effect on PLK. These results and the clinical consequences of such interactions are discussed and compared with results of previous studies.

Effect of long-term treatment with insulin and/or acarbose on glomerular basement membrane thickening in alloxan-diabetic rats

Acarbose is a competitive inhibitor of the intestinal alpha-glycosidases, that can delay absorption of intestinal carbohydrates causing their malabsorption. In the present paper we studied the effects of insulin, acarbose and their association on glomerular basement membrane thickening in alloxan-diabetic rats. Twenty-five male and female Wistar rats, approximately 3 months old at the beginning of the experiment, were assigned randomly to each of five experimental groups: normal control rats, alloxan-diabetic control rats, alloxan-diabetic rats treated with acarbose, alloxan-diabetic rats treated with insulin, and aloxan-diabetic rats treated with insulin plus acarbose. Alloxan was administered in a single iv dose of 42 mg/kg body weight. Insulin was given subcutaneously at doses of 18 to 30 IU/kg corrected daily on the basis of glycosuria and ketonuria. Acarbose was given mixed with rat chow in a dose of 50 mg/100 g chow. Body weight, water and food intake and diuresis, as well as blood and urine glucose were determined after 1, 3, 6, 9, and 12 months of treatment. Glomerular basement membrane (GBM) thickening was determined by electron microscopy at the same times. Clear clinical and laboratory signs of severe diabetes, with blood glucose levels above 200 mg/dl and urine glucose above 3000 mg/dl, were observed in all alloxan-diabetic control rats, in all periods of follow-up, whereas administration of insulin or acarbose reduced the blood glucose levels of treated groups. The most satisfactory control of blood and urine glucose was observed in animals treated with both insulin and acarbose. However, diarrhea was observed in diabetic rats treated with acarbose associated or not with insulin, GBM thickening was correlated with age in all groups. Beginning at six months after diabetes induction, the GBM of untreated diabetic rats was significantly thicker (mean + 4.446 + 0.45 mm) than that of normal rats (2.977 + 0.63 mm). Both insulin and acarbose prevented GBM thickening and their combination induced thickening similar to the age-dependent thickening observed for normal rats of the same age. We conclude that acarbose when combined with insulin may be a good option in the control of diabetes and its renal complications.

Effect of various diuretics on membrane voltage of macula densa cells. Whole-cell patch-clamp experiments.

The tubuloglomerular feedback mechanism is inhibited by diuretics such as furosemide. For the macula densa (MD) cells similar transport systems, as present in thick ascending limb (TAL) cells, have been suggested. To examine this further, membrane voltages (Vm) of MD cells were recorded with the fast or slow whole-cell patch-clamp method. The effects of diuretics on voltages and the conductance properties of these cells were examined. Vm of MD cells measured with the whole-cell patch-clamp method were as high as those in TAL cells: -72 +/- 1 mV (n = 21). An increase in the extracellular K+ concentration by 15 mmol/l depolarized Vm of MD cells by 11 +/- 1 mV (n = 18). Ba2+ (1 mmol/l) reversibly depolarized MD cells by 10 +/- 2 mV (n = 10). Thus, MD cells possess a K+ conductance that could allow for the recycling of K+ taken up by the Na(+)-2 Cl(-)-K+ cotransporter. MD cells hyperpolarized reversibly upon addition of the loop diuretics furosemide, piretanide and torasemide, whereas muzolimine and hydrochlorothiazide, neither one acting on this cotransport system in other preparations including the TAL, had no effect on Vm. MD cells most likely possess the same cotransport system as the TAL cells, which drives NaCl reabsorption in the TAL and serves as sensor for the tubular NaCl concentration in MD cells.

Clinicopharmacological reappraisal of the potency of diuretics.

From a clinicopharmacological standpoint, the urinary excretory potency of diuretics should be assessed comparatively on the basis of the changes in 24-hour natriuresis, with respect to 24-hour natriuresis after placebo, caused by single oral doses administered to healthy adult subjects who are in habitual and steady-state external sodium balance. The potency of various formulations of loop (e.g., furosemide), of early distal tubular (e.g., the thiazides), and of potassium-retaining diuretics, as well as of several combinations of diuretics, has been evaluated in a series of studies. Two formulations of loop diuretics (muzolimine 20 mg and torasemide 2.5 mg) are definitely nondiuretic. The majority of the other formulations of loop diuretics studied are, in general, comparatively less potent than most of the common formulations of early distal tubular diuretics studied. As a general rule, most common formulations of early distal tubular diuretics are at least not less potent than the majority of common formulations of loop diuretics. Hydrochlorothiazide 25 mg and furosemide 80 mg have similar potencies. Loop diuretics increase mean renal sodium output strikingly within the first few (0-6) hours after dosing, but this forced excretion is followed by a rebound with respect to postplacebo mean urinary sodium flow; the rebound usually takes place between 6 and 24 hours after dosing. However, no rebound in mean urinary sodium flow occurs during the 24 hours following a single dose of a distal tubular diuretic; these substances increase urinary sodium excretion with lower maximal intensity but more protractedly than loop diuretics.(ABSTRACT TRUNCATED AT 250 WORDS)

Central hemodynamic effects of diuretic therapy in chronic heart failure.

In chronic heart failure diuretic drugs improve central hemodynamic variables and cardiac pumping secondary to altered plasma and extracellular volumes; humoral markers of these changes include increased plasma renin and aldosterone levels. The latter increases are maximal over the first week but decline with chronic therapy. The plasma alpha-ANP levels show a reciprocal effect; these data are compatible with a rapid contraction of the plasma volume which is sustained during chronic therapy. The acute hemodynamic actions of diuretic agents reflect both immediate and direct vascular actions and also effects secondary to diuresis (volume redistribution). At rest substantial reductions in pulmonary "wedge" pressure (-29%), with a consequent fall in cardiac output (-10%), are described. Total systemic vascular resistance initially increases but "reverse autoregulation" over subsequent weeks returns this elevation gradually towards control values. Tolerance to these initial hemodynamic effects does not occur with maintained therapy; moreover, echocardiographic markers of contractility and exercise capacity may increase. The early venodilator effects of diuretic drugs can be attributed to prostaglandin release and the initial pressor actions to activation of the renin angiotensin system; these vascular actions may have limited relevance to long-term beneficial effects on hemodynamics. Direct pulmonary vasodilation and improved pulmonary compliance remain an interesting finding. Although most patients are both symptomatically and hemodynamically improved at rest, the actions during exercise are more varied. Some individuals with severely impaired left ventricular function show little hemodynamic improvement, whereas those with milder dysfunction usually benefit; in the main this is probably related to the latter being on a steeper cardiac function curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Muzolimine: renal site of action and interaction with probenecid in humans.

Muzolimine (60 mg, administered orally) was administered to eight healthy volunteers, under conditions of altered fluid load, to elucidate its renal site of action. The duration of action and the effect of probenecid pretreatment on muzolimine response was also investigated. Muzolimine had a rapid onset of action, with the diuresis complete within 4 hours after dosing. At peak natriuresis, under hydrated conditions, fractional excretion of free water remained unaltered (9.72% +/- 0.59% versus 9.07% +/- 0.44%; difference not significant) but was accompanied by a significant increase in the delivery of sodium out of the proximal tubule, as measured by fractional excretion of lithium (22% +/- 2% to 31% +/- 1%; p less than 0.01). The fraction of sodium reabsorbed in the distal tubule also decreased from 94% +/- 1% to 67% +/- 1% (p less than 0.001) of the delivered load. The fractional reabsorption of free water during hydropenia decreased after muzolimine (5.63% +/- 0.26% to 2.00% +/- 0.81%; p less than 0.05). Pretreatment with probenecid resulted in a prominent decrease in urinary sodium excretion (246 +/- 25 mmol/24 hr for muzolimine alone 161 +/- 24 mmol/24 hr for muzolimine and probenecid; p less than 0.01). These findings suggest that muzolimine has a major site of action in the medullary portion of the thick ascending limb of Henle with additional inhibitory activity on the proximal tubule. It is likely that the active secretion of one or more of the acidic metabolites of muzolimine, by way of the probenecid sensitive organic acid pathway, is responsible for mediating the renal actions this basic drug.

The effects of muzolimine and urine from muzolimine-treated rats on Na+K+Cl- cotransport in Madin-Darby canine kidney cells.

Muzolimine is a loop diuretic with an original chemical structure devoid of the acidic or sulfonamide group known to be necessary for an interaction with Na+K+Cl- cotransport. We studied the effects of urine from muzolimine-treated rats on the Na+K+Cl- cotransport-dependent 86Rb influx in MDCK cells. Na+K+Cl- cotransport was inhibited by urine obtained 15 min (42% inhibition) and 60 min (49% inhibition) after muzolimine injection (50 mumol/kg i.v.). Muzolimine itself was not detectable in the urine. Probenecid (100 mumol/kg i.v.) suppressed both the diuretic effect of muzolimine and the inhibition of Na+K+Cl- cotransport by urine from muzolimine-treated rats. These results suggest that the diuretic effect of muzolimine is due to the metabolism of muzolimine into an active compound which inhibits Na+K+Cl- cotransport after its secretion into the tubular lumen via a proximal pathway. The direct effect of muzolimine on Na+K+Cl- cotransport in MDCK cells was also tested: surprisingly, the inhibition of 86Rb influx was significant in the presence of muzolimine (IC50 = 1.44 microM). We show that this effect was due to the metabolism of muzolimine by these cells into an active compound.

Muzolimine-induced severe neuromyeloencephalopathy: report of seven cases.

We report on 7 patients (2 women, 5 men) with chronic renal failure, who developed under a high dosage of the new diuretic muzolimine (range 240 to 1440 mg per day) fatal neuromyeloencephalopathy. Clinical neurophysiological and neuroradiological findings and finally neuropathological studies in 2 patients resembled those found in vitamin-B12-deficiency-syndrome with a predominant affection of the spinal posterior column and the corticospinal tracts. The first neurological symptoms like paraesthesia, severe hyperpathia of the legs and mild to heavy spinal ataxia occurred after an average time of treatment of 78 days and a mean dosage of 52 g. The most progressive neurological deficits like severe tetraspastic paresis, were seen only in the nondialytic renal insufficient group (3 patients), while the others had a more benign course of the disease. This lead to the hypothesis of a partially dialysable toxic metabolite of muzolimine. After a follow-up of more than 2 1/2 years no significant recovery was seen in these cases.

Comparison between the cardiac effects induced by muzolimine and furosemide in guinea-pig atria.

Muzolimine (10-500 microM) induced a concentration-dependent reduction of both the contractile force and frequency in spontaneously beating atria and in electrically driven left atrium from reserpine-treated guinea pigs. This negative inotropic response was unaffected by the addition of atropine to the perfusion fluid, and it was highly sensitive to changes in external Ca2+ concentration. Both in spontaneously beating and in electrically driven atrium, muzolimine (50-400 microM) antagonized, in an apparently competitive manner, the increase in contractile force induced by cumulative addition of CaCl2 (0.68-9.59 mM) to the bathing fluid. Muzolimine (50-100 microM) reduced the inotropic response to low (5-30 nM), but not high (50-100 nM) concentrations of Bay K 8644, a calcium-channel agonist. The inotropic effects of 8-phenyltheophylline and of ouabain were antagonized by muzolimine (10-100 microM) in a noncompetitive manner, while the response to noradrenaline was not altered. Similar to muzolimine, verapamil at a concentration suitable to block calcium channels inhibited, in a noncompetitive way, the inotropic effect induced by 8-phenyltheophylline and by ouabain without altering the contractile response to noradrenaline. Furosemide (10 and 100 microM) did not influence the contractile force or the frequency of spontaneously beating atria, nor the inotropic effect induced by CaCl2, 8-phenyltheophylline, ouabain, or noradrenaline. These results indicate that the influence of muzolimine on guinea-pig atria originates from an inhibition of Ca2+ influx into cardiac cells and that furosemide does not mimic the effect of muzolimine at this level.

[Mechanism of action of the diuretic effect of muzolimine]. / Mécanisme d'action de l'effet diurétique de la muzolimine.

Muzolimine is a diuretic which has been proposed in the treatment of hypertension. Muzolimine shared both the high ceiling effect of loop diuretics and the long duration of action of thiazides but has a chemical structure different from those of other loop diuretics. It may act as a prodrug and an active metabolite present in the urine may inhibit NaCl reabsorption in the Henle's loop. We studied the effect of urines of piretanide and muzolimine treated rats on Na+K+Cl- cotransport in renal cells in culture (MDCK). In the presence of ouabain (0.5 mM), the Na+K+Cl- cotransport measured by 86Rb influx, represented 92 p.100 of the total 86Rb influx (6.16 +/- 1.12 nmol 86Rb/min/mg prot, n = 10). Both diuretics were administered i.v. to rats where they induced marked diuresis. Excreted urine (dilution to 1/100) was tested for cotransport inhibition. After piretanide (27 mumol/kg) the urine inhibited the Na+K+Cl- cotransport in MDCK cells (72% and 41% inhibition at the 15th and 45th minutes after diuretic injection). After muzolimine (50 mumol/kg), urines also inhibited Na+K+Cl- cotransport but the effect was slower in onset and more prolonged (42% and 49% inhibition at the 15th and 60th min). Diuretic effects in vivo and Na+K+Cl- cotransport inhibition in vitro by the urine developed parallel for both diuretics. Probenecid (100 mumol/kg) suppressed simultaneously the diuretic effect of muzolimine and the Na+K+Cl- cotransport inhibition by the urine of muzolimine treated rats. Our results suggest that muzolimine acts as a prodrug. Its active metabolite is secreted into the tubular lumen through a probenecid sensitive pathway and inhibits, like other loop diuretics, Na+K+Cl- cotransport.

[Neuromyeloencephalopathy caused by high-dose muzolimine medication in patients with renal failure]. / Neuromyeloenzephalopathie (NME) unter hochdosierter Muzolimin-Medikation bei niereninsuffizienten Patienten.

We report on 7 patients suffering from chronic renal failure (2 females, 5 males; aged 35-75 (phi 53.5) years) who showed severe neuromyeloencephalopathy (NME) after high doses of a new Henle's loop diuretic, Muzolimine. The temporal and phenomenological development of these systems was strikingly parallel. The neurological deficit was revealed on neurophysiological, neuroradiological and in 2 cases on neuropathological tests (gross demyelinisation of the posterior column, mainly of the fasciculus gracilis, less in the lateral corticospinal tract and in some spinal roots). The critical drug dose for first neurological impairment was 52 g on average; at this point the patients had been treated for 78 days. The maximal daily dose was 1.440 mg. Dominant clinical features were pallhypaesthesia, ataxia, signs of peripheral neuropathy in combination with hyperreflexia and progressive para- to tetraspastic paresis. Constellation of symptoms, course of disease and findings of additional investigations, especially those of neuropathology, very much resemble Vitamin B12 deficiency and SMON-(Subacute Myelo Optic Neuropathy) syndrome. The rare entity of Muzolimine-NME is discussed in respect to other endogenous and exotoxic neuromyelopathies. We present the hypothesis of a toxic, partially dialysable metabolite of Muzolimine.

[Neurotoxic action of muzolimine at high doses in uremic patients. Observation of a group of 29 subjects]. / Azione neurotossica della muzolimina ad alte dosi in pazienti uremici. Osservazione su un gruppo di 29 soggetti.

We report the results of a study on 29 patients affected by renal chronic insufficiency and treated with high doses of muzolimine. From our data it results that to the muzolimine is probable due a neurological syndrome very similar to combined sclerosis. Up today, it is not possible to know how and where the muzolimine develops its neurotoxic effect.

Platelet cytosolic free calcium concentration in hypertension associated with early stage kidney disease.

Chronic hypertension accompanying early stage kidney disease is characterized by increased vascular resistance, but the underlying processes responsible for the enhanced vascular tone are unclear. We studied free calcium levels in blood platelets with the fluorescent dye quin-2. Platelets have many features in common with vascular smooth muscle cells. The cytosolic calcium concentration in platelets was elevated in 27 renal hypertensive patients, who were compared with 12 normotensive subjects (P less than 0.001). There was a close correlation between the free calcium level and mean blood pressure (r = 0.88, P less than 0.001). Short-term antihypertensive treatment with a calcium entry blocker or a diuretic resulted in a significant reduction in cytosolic calcium (P less than 0.05), and this correlated with the fall in blood pressure (r = 0.95, P less than 0.001). These data suggest an integrative contributory role of calcium in the pathophysiology of hypertension accompanying early stage kidney disease.

Effects of muzolimine on the late distal tubule of necturus kidney.

The in vivo effects of the diuretic muzolimine were studied in the late distal tubule of the amphibian Necturus. Conventional and ion-selective microelectrodes were used to determine basolateral membrane potential, intracellular Cl- activity and luminal activities of Cl- and K+. Muzolimine depolarized basolateral membrane potential by about 30 mV in 1 min, in a reversible fashion. We attributed this depolarization to blockade of a K+ conductance, because the effects of muzolimine and barium on the highly K-selective basolateral membrane were not additive. In addition, muzolimine elicited a reversible increase of intracellular Cl- activity from 7.5 +/- 0.5 to 14.5 +/- 2.6 mM (concomitant to the basolateral membrane potential depolarization) and of luminal activities of Cl- from 12.4 +/- 1.5 to 22.3 +/- 2.5 mM, within approximately 1 min; both disturbances relaxed toward control values after withdrawal of the diuretic. That muzolimine increases Cl- activity in both the cell and the lumen of the late distal tubule (Cl- is accumulated in these compartments), indicates that retention of Cl- results from hindrance of the basolateral exit step rather than of apical Cl- uptake. Inasmuch as muzolimine failed to increase the luminal activity of K+, Cl- is believed to accumulate in the lumen as NaCl, not KCl.

Long-term treatment of essential arterial hypertension with nitrendipine.

The efficacy and safety of long-term treatment with oral nitrendipine were evaluated in 34 patients with essential arterial hypertension. Nitrendipine alone significantly lowered systolic and diastolic blood pressure levels in 28 patients who completed the preliminary four-week dose-setting phase. Twenty-one patients completed the one-year treatment. Blood pressure control was maintained by nitrendipine alone in 11 patients. Ten patients not adequately controlled at the end of the dose-setting phase were successfully treated with nitrendipine combined with acebutolol or muzolimine. It is concluded that nitrendipine is a promising calcium antagonist for the treatment of arterial hypertension.

The diuretic effect of muzolimine.

Muzolimine produces a diuresis in the loop of Henle. Unlike other diuretics of this kind, the effect of muzolimine is slow and its action is long lasting. The present study was designed to examine the mechanism of action of muzolimine in isolated in vitro perfused rabbit cortical thick ascending limb segments (cTAL). In a first series it was confirmed that muzolimine itself in a concentration of up to 10(-4) mol/l had no effect on the equivalent short circuit current (Isc), corresponding to the rate of active NaCl reabsorption, in cTAL segments neither from the lumen nor from the bath side. In a second series of experiments, muzolimine was administered intravenously (70 mumol/kg) to male Wistar rats, and the clearances of inulin, Na+ and K+ were calculated. Muzolimine, after a lag phase of 5 to 20 min caused a marked diuresis and natriuresis, but had only a minor effect on glomerular filtration rate and on K+ excretion. The urines of these animals were diluted with isotonic saline, and were examined in rabbit cTAL segments for their ability to block Isc. It was found that the urines of the antidiuretic period were devoid of effects, whereas the diuretic urines inhibited Isc strongly down to a dilution of 300 times when they were perfused in the lumen. The same diluted urines had no effect from the bath side. In a third series, probenecid (100 mumol/kg) was administered i.v. to rats undergoing a muzolimine induced saluresis. It was found that probenecid inhibited the diuresis and saluresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the Cl-/NaCO3- anion exchanger by xipamide in human red blood cells.

The mechanism of action of classical loop diuretics of the 2- or 3-amino-5-sulfamoylbenzoic acid and (aryloxy)acetic acid families involves competition with chloride for a common site on the (Na+, K+, 2Cl-) co-transport system. However this is not the mechanism of action of some high-ceiling diuretics like muzolimine, MK 473, xipamide, indapamide and clopamide, which are not carboxylic acids. We evaluated three of these latter diuretics (xipamide, muzolimine and clopamide) for their inhibitory effects on five ion transport systems in human red blood cells: (i) Cl(-)-dependent (Na+, K+) co-transport, (ii) (NaCO3-/Cl-) anion exchanger, (iii) (Cl-, K+) co-transport, (iv) Na+, K+ pump and (v) Na+: Li+ counter-transport; and on one ion channel the Ca2+-dependent, K+ channel. All erythrocyte transport pathways were resistant to the three diuretics studied (IC50 of 10(-3) M or higher) with one remarkable exception, the (NaCO3-/Cl-) anion exchanger. This transport system was inhibited by xipamide (IC50 of 2.5 +/- 0.4 X 10(-5) M, mean +/- S.D. of five experiments) and less potently by muzolimine (IC50 of 1.1 +/- 0.3 X 10(-4) M, mean +/- S.D. of three experiments). Clopamide only inhibited the anion exchanger at high concentrations (IC50 of about 10(-3) M). Xipamide, the most potent diuretic in this test, was at least one order of magnitude more active than furosemide, ethacrynic acid, hydrochlorothiazide and amiloride. Inhibition of the anion carrier could be involved in the diuretic action (inhibition of CO2-stimulated NaCl absorption in the TAL) and/or in the antihypertensive action (inhibition of net NaCO3- influx and secondarily of Ca2+ influx through Na+: Ca2+ exchange in vascular smooth muscle cells of xipamide).