Calcium Inhibitors Research Articles

Modulation of Pea Membrane β-Glucan Synthase Activity by Calcium, Polycation, Endogenous Protease, and Protease Inhibitor

beta-Glucan synthase activity in plant membranes can be markedly altered by a multiplicity of apparently unrelated factors. In pea epicotyl membranes it is enhanced by low and inhibited by high concentrations of added Ca(2+), trypsin or soluble pea protease. Ca(2+) stimulates preexisting synthase activity, particularly in the presence of polycations (spermidine), but protease treatments activate and, with time, inactivate synthase zymogen. Endogenous pea protease activity is also associated with washed pea membrane and appears to be responsible for the decay observed with time in the beta-glucan synthase activity. Endogenous pea protease activity is inhibited by thiol inhibitors, e.g. iodoacetamide and Hg(2+), and by a heat-stable peptide, molecular weight approximately 10,000, that is found in supernatants of pea extracts. These protease inhibitors have the capacity to protect beta-glucan synthase activity from denaturation or its zymogen from activation due to endogenous or added protease activity. Evidence is described which supports the proposal that 1,4-beta-glucan synthase is destroyed and possibly converted to 1,3-beta-glucan synthase activity by protease action, and that the latter may then be greatly enhanced by Ca(2+) and polycations.

7 Calcium Channel Blocking Drugs

This chapter discusses calcium channel blocking drugs. Many new drugs have been developed for the treatment of cardiovascular disorders. One of the most important discoveries has been the existence of drugs that inhibit the entry of calcium ions into cells by blocking the voltage-dependent calcium channels. Because drugs may inhibit the availability of calcium by blocking voltage-dependent calcium channels, by stimulating the efflux of calcium from the cell or by stimulating the uptake of calcium into storage sites, the term calcium antagonist is clearly a rather imprecise one in describing the mechanism of action of such drugs. The use of other terms, such as calcium entry blocker, calcium inhibitor, or slow channel blocking drug, is similarly inexact. Drugs that act through the inhibition of the inward slow calcium current by preventing the passage of Ca 2+ ions through the calcium channel in the cell membrane are best described as calcium channel blocking drugs. These calcium channel blocking drugs form a diverse group of substances.

The protective effect of calcium inhibitors and of captopril on the renal microcirculation during reperfusion.

Reperfusion injury is increasingly recognized as a key factor in the development of posttransplant acute tubular necrosis. Previous studies have shown that addition of the calmodulin inhibitor trifluoperazine (TFP) to Collins' flush solution protected the cortical microcirculatory integrity and dramatically improved renal viability after transplantation. The present report describes the protective effect(s) of TFP in the course of reperfusion injury. Twenty mongrel dogs underwent bilateral nephrectomy; in each instance, the left kidney was flushed immediately with 250 ml of cold Collins' solution, and the right kidney was flushed with the same solution containing TFP, 5 mg/L. After 48 and 72 hr of preservation, each kidney was connected through silastic shunts to the femoral vessels of another dog. The mean renal blood flow (RBF) immediately after reperfusion was 2.2 ml/g/min and 1.7 ml/g/min in the left and right kidneys, respectively, and was similar to mean RBF measurements prior to nephrectomy. After 15 min of reperfusion, there was a sharp decrease in mean RBF in the Collins' flushed kidneys, which persisted after 60 min of reperfusion (0.37 ml/g/min). In contrast, there was only a mild decrease in mean RBF in the TFP-flushed kidneys (1.27 ml/g/min). A partial explanation for the favorable effect of TFP may be related to the inability of the ischemic cell to handle the increased calcium load associated with reperfusion (calcium paradox). In a test of this possibility, 0.5 mg/kg of verapamil, a calcium channel blocker, was infused during reperfusion. No beneficial effects of this drug were noted in either Collins' or TFP-flushed kidneys (n = 10). However, when 1.25 mg/kg of captopril, an angiotensin-converting enzyme inhibitor, was infused at the time of reperfusion, a dramatic amelioration of the reperfusion injury occurred in the Collins' flushed kidneys (1.2 ml/g/min) (n = 10). Taken together, these data suggest that the damage to cold-preserved kidneys flushed with Collins' solution alone may occur at the time of actual reperfusion. Such reperfusion damage is ameliorated by TFP and captopril. The known relationship between calcium and the effect of angiotensin on the vascular smooth muscle cell may explain in part the protective role of calcium inhibitors placed in preserved kidneys prior to reperfusion.

Permeability changes in the egg-shell of hookworms during development and eclosion.

The development and hatching of Ancylostoma ceylanicum and Ancylostoma tubaeforme eggs in a range of potentially inimical solutions has been examined using time-lapse video micro-recording techniques. In all compounds tested the larvae developed to the active pretzel stage. The inception of activity resulted in an increase in shell permeability to water and small molecules. In anthelmintics of molecular weight (Mr) 400 development continued normally until about the time that control eggs hatched, when there was a marked decrease in larval activity and eclosion did not occur. The addition of a low Mr surface-active agent potentiated the effect of the anthelmintic and caused larval activity to be lost earlier. The calcium inhibitor lanthanum chloride affected larval development rather than eclosion. Large Mr compounds did not affect either development or hatching and the results suggest that the egg-shell may act as a form of sieve, allowing the essential oxygen to reach the developing larva while excluding large potentially dangerous molecules. No appreciable differences were found between the two species of hookworm examined.

Acute and Chronic Effects of a New Calcium Inhibitor, Nicardipine-on Renal Hemodynamics in Hypertension

Renal hemodynamics and natriuresis were studied in 10 hypertensive patients without renal failure, 2 and 4 h after oral intake of 30 mg nicardipine; then, nicardipine was given at a dose of 30 mg three times a day and the hemodynamic study was repeated on the 6th day (2 h after the morning dose). The first dose of nicardipine produced an increase in renal blood flow (from 888 +/- 45 to 999 +/- 59 ml/min 1.73 m2, p less than 0.01) and a decrease in renal vascular resistances (from 0.16 +/- 0.01 to 0.12 +/- 0.01 arbitrary unit, p less than 0.05). Glomerular filtration rate did not change and the decrease in filtration fraction was not significant. Sodium excretion increased markedly during the first 2 h (from 0.17 +/- 0.04 to 0.29 +/- 0.06 mmol/min, p less than 0.05). On the 6th day renal vascular resistances and filtration fraction remained lowered whereas glomerular filtration rate was unchanged. Nicardipine did not produce any significant alteration in plasma renin activity and plasma aldosterone after acute or chronic administration. These results confirm the potent renal vasodilatory effect of nicardipine; glomerular filtration rate was not significantly altered whereas renal blood flow and filtration fraction returned to normal levels. An early and transient natriuretic effect was observed after the first dose of nicardipine, and body weight showed a significant decrease during the study indicating that no sodium retention was induced by nicardipine.

Oosporogenesis by Lagenidium giganteum: induction and maturation are regulated by calcium and calmodulin.

Induction and maturation of the sexual stage (oospores) of the facultative mosquito parasite Lagenidium giganteum (Oomycetes: Lagenidiales) are complex developmental processes regulated by calcium-dependent events. Use of developmentally synchronized cultures of L. giganteum allowed stage-specific disruption of calcium metabolism. A calcium chelator (EGTA), an ionophore (chlortetracycline), and inhibitors of the calcium-binding protein calmodulin (dibucaine, trifluoperazine, chlorpromazine) disrupted several discrete developmental steps associated with oosporogenesis: induction of antheridia, gametangial fusion, meiosis, oospore wall formation, and subsequent spore maturation. Extracellular calcium is necessary for oosporogenesis to proceed normally and under some conditions magnesium has a synergistic effect with calcium on oospore induction. Results are discussed in relation to calcium mediation of fusion events in a number of model membrane and biological systems.

Effect of low calcium and protease inhibitors on synapse elimination during postnatal development in the rat soleus muscle

The mechanisms controlling the reorganisation of synaptic inputs to developing skeletal muscle fibres was studied using electrophysiological and histological methods. In the developing rat soleus muscle there is a rapid reduction of polyneuronal innervation between 9 and 12 days. Reducing the local concentration of calcium by applying chelating agents such as EGTA or BAPTA in vivo to 9-day-old rat soleus muscles over a period of 3 days slowed the rate of elimination of polyneuronal innervation. It was established that the reduction of calcium induced by EGTA or BAPTA was not sufficient to produce a detectable reduction in neuromuscular activity. The possibility that a calcium-dependent enzyme such as CANP may play a role in synapse reorganisation was therefore tested. Local application of inhibitors of calcium-activated neutral protease (CANP), leupeptin or E-64, to 9-day-old rat soleus muscles over 3 days had similar effects to those of EGTA or BAPTA, i.e. the elimination of polyneuronal innervation that usually takes place was much slower. Since the inhibition of thiol proteases had similar effects on synapse elimination as a reduction of calcium concentration, it is concluded that CANP is important in the reorganisation of the developing neuromuscular junction.

The effect of calcium antagonists and inhibitors of secretory processes on auxin-induced elongation and fine structure ofPisum sativum stem segments

The treatment of dark grown pea stem segments with chelators of divalent cations (EGTA, EDTA, CTC), various Ca2+ antagonists (LaCl3, A-23187, verapamil) and inhibitors of secretory processes (monensin, CB) reduced elongation in the presence of indole-3-acetic acid (IAA). Generally the inhibition increased with increasing concentrations of the substances. The timing of the responses can be correlated with maximum auxin-stimulated secretion of cell wall material. Examination of cell ultrastructure showed that changes in dictyosome activity could explain a reduced deposition of cell wall material and so cause inhibition of elongation.

TRH and GRF stimulate release of growth hormone through different mechanisms.

Thyrotropin-releasing hormone (TRH) is an effective stimulator of growth hormone (GH) release from cultured adenohypophysial cells of chronically hypothyroid rats in vitro. The present study explored the question of cAMP and calcium mediation of the GH-stimulatory effect of TRH in this system. A maximally stimulatory concentration of TRH was added together with various concentrations of human GH-releasing factor 40 (hGRF-40) whose action is cAMP mediated, or of dibutyryl cAMP (DBcAMP), to primary monolayer cultures of adenohypophysial cells from thyroidectomized rats. The GH responses to the combined addition of TRH with all doses of GRF or DBcAMP were fully additive, causing parallel elevations of the dose-response curves. Whereas the GH response to maximally effective concentrations of hGRF-40 and DBcAMP, added together, was not greater than that to either secretagogue alone, the inclusion of TRH increased the response to a new Emax. The calcium inhibitors, verapamil, EGTA, and CoCl2, markedly suppressed basal GH release and virtually completely blocked the GH response to TRH, suggesting calcium mediation. In chronically hypothyroid, urethan-anesthetized rats, the in vivo effect of the combined administration of maximally effective doses of TRH and GRF on plasma GH levels was also additive. These findings indicate that TRH stimulates GH release in adenohypophysial cells of hypothyroid rats by a cAMP-independent, calcium-dependent mechanism.

Multiple calcium-activated neutral proteinases (CANP) in mouse retinal ganglion cell neurons: specificities for endogenous neuronal substrates and comparison to purified brain CANP

Calcium-activated neutral proteinases (CANPs) and their specificities for axonally transported proteins were studied within intact axons of mouse retinal ganglion cell (RGC) neurons in vitro. Two CANP activities with markedly different properties were identified. CANP B, at endogenous calcium levels, selectively cleaved the 145,000 Da (145 kDa) neurofilament protein subunit to yield 143 and 140 kDa neurofilament proteins that are also major constituents of the axonal cytoskeleton. This process represents a posttranslational modification of the neurofilament protein subunit rather than the initial step in its degradation (Nixon et al., 1982, 1983). A second calcium-activated neutral proteinase activity, CANP A, appeared only when calcium levels in the incubating medium were 100 microM or higher. CANP A degraded most proteins in RGC axons but acted considerably more rapidly on high-molecular-weight species. In particular, a 290-320 kDa protein in the Group IV (SCb) phase of axoplasmic transport was degraded 3 X faster than other major axonal proteins, including neurofilament proteins and fodrin. When maximally expressed, CANP A activity represented an enormous proteolytic potential in RGC axons--more than 50% of the total axonal content of proteins larger than 60 kDa could be hydrolyzed within 5 min. The calcium requirements, inhibitor profile, and substrate specificity of CANP A were similar to those of mCANP, the major CANP of mouse brain purified to homogeneity, suggesting that these enzymes may be the same or highly related proteins. The existence in a single neuron type of two CANP activities with markedly different substrate specificities and enzymatic properties emphasizes the possible functional diversity of calcium-activated neutral proteinases in neurons. These functions include the posttranslational modification, as well as degradation of neuronal proteins.

The mode of vasorelaxant action of 2-aminoisoquinoline, 1.3 (2H.4H)-dione, a novel 'intracellular calcium inhibitor'.

In rabbit aorta, pretreatment with 2-aminoisoquinoline, 1.3 (2H.4H)-dione (AQ, 10(-5) M and 10(-4) M) shifted the concentration-response relationship to noradrenaline (NA, 10(-9) M to 10(-4) M) in a parallel manner whereas the agent (10(-4) M) failed to affect the response to potassium and only slightly depressed Ca2+-induced contractions in a Ca2+-free medium in the presence of K+ (40 mM). Ca2+-entry blockers such as nifedipine and diltiazem (10(-6) M and 10(-5) M) had very weak or no apparent effects on the response to NA but markedly attenuated or abolished the K+- and Ca2+-induced contractions. Following incubation of tissues for 15 min in a Ca2+-free medium with low EGTA (0.01 mM) and methoxyverapamil (D600, 10(-5) M), NA (3 X 10(-7) M) caused a phasic (transient) contraction and the subsequent application of Ca2+ (2mM) resulted in a tonic contraction. This NA-induced, Ca2+-dependent, D600-insensitive contraction was inhibited by AQ (10(-5) M and 10(-4) M) in a concentration-dependent manner. This suggests that the inhibitory action of AQ may be related to Ca2+ entry through specific receptor activated pathways. Following incubation of tissues for 30 min in a Ca2+-free medium with high EGTA (2.0 mM), NA (10(-5) M) caused a contraction of rabbit aorta which is dependent upon release of intracellular Ca2+, but the response was 50% to 60% less than that in a normal medium. This contraction was inhibited by AQ (10(-5) M and 10(-4) M) and nitroglycerin (10(-5) M) but not by nifedipine or diltiazem. The inhibitory action of combined treatment with AQ and nitroglycerin (10-5 M) on the response to NA was not different from that of either agent alone. 5 These results suggest that AQ may have inhibitory actions on the release of intracellular Ca2+ and also on Ca2+-entry through D600-insensitive, receptor-activated Ca2+ pathways in rabbit aorta.

Acute and Chronic Effects of a New Calcium Inhibitor, Nicardipine, on Renal Haemodynamics in Hypertension

s: Groupe Hypertension de la Société Française de Cardiologie, Journées de L'Hypertension Artérielle Faculté Pitié-Salpêtrère, Paris: Oral Papers: PDF Only

Interest of a New Calcium Inhibitor, Nicardipine, in Severe Renal Insufficiency

s: Groupe Hypertension de la Société Française de Cardiologie, Journées de L'Hypertension Artérielle Faculté Pitié-Salpêtrère, Paris: Oral Papers: PDF Only

ATP-dependent [ 3H]met-enkephalin uptake by bovine adrenal chromaffin granule membrane

Uptake of [ 3H]Met-enkephalin by purified chromaffin granule membrane isolated from bovine adrenal medulla was investigated. Addition of a single divalent cation, such as Mg 2+ or Ca 2+, did not affect the uptake. The presence of 3 mM ATP in the incubation medium stimulated the uptake to two times that of the control. The same stimulation effect was also found in the presence of ATP plus a single divalent action. The ATP-dependent uptake reached its half-maximal level within 5 min after initiation of the reaction at 25°C, and reached a plateau within 10 min. The apparent K m for [ 3H]Met-enkephalin uptake by the chromaffin granule membrane was about 2.3 × 10 −6 M. GTP, UTP, UTP and ADP did not stimulate enkephalin uptake. Several calcium inhibitors such as trifluoperazine, verapamil and 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octylester (TMB-8) markedly inhibited enkephalin uptake. The inhibition exceeded 80%, although these chemicals inhibited Ca 2+-ATPase activity in chromaffin granule membrane only 30% under the same conditions. Ethyleneglycol-bis-(β-aminoethylether)N,N′-tetraacetic acid (EGTA), at 3 mM also inhibited uptake about 30%. The results indicate that uptake of Met-enkephalin by chromaffin granule membrane ghosts was driven not only by ATPase activity, but also by some other Ca 2+-ATP-mediated mechanism(s).

Management of Hypertension in Patients with Obstructive Airway Disease

T he past three decades have seen remarkable progress in the development of effective antihypertensive agents. In no other area of drug research, except perhaps the antibacterials, has there been such notable success. The availability of several new classes of antihypertensive agents has allowed for effective control of hypertension without some of the serious side effects, including mental changes and postural hypotension, that were seen with older agents. Currently available antihypertensive agents may be classified into two groups depending on whether they act by decreasing cardiac output or by lowering the peripheral vascular resistance (Table 1). The most important agents that act primarily by decreasing cardiac output are the �3-adrenergic antagonists. Agents that act primarily to reduce peripheral vascular resistance include the a-adrenergic blocking agents, the central a-adrenergic inhibitors, the direct smooth muscle relaxants, and the angiotensin-converting enzyme inhibitor captopril. Calcium inhibitors are listed in Table 1 as direct smooth muscle relaxants, although verapamil and diltiazem also slow intracardiac conduction and may decrease heart rate. While not yet approved as antihypertensive agents in this country, they are commonly used for this purpose. Diuretics decrease both cardiac output and peripheral resistance. 13-ADRENERGIC ANTAGONISTS

Systemic and Renal Hemodynamic Effects of Single Oral Doses of Felodipine in Patients with Refractory Hypertension Receiving Chronic Therapy with β-Blockers and Diuretics

In 12 patients with primary hypertension (World Health Organization stage 2) inadequately controlled by chronic standard triple therapy, hydralazine was replaced by felodipine, a new vasodilating dihydropyridine derivative, and the acute effects of the drug on central and renal hemodynamics were monitored. Following baseline measurements, an oral solution of felodipine (0.075-0.1 mg/kg) was given. Fifteen minutes after intake of felodipine, a significant hypotensive response was observed, and the maximal response (23% reduction of mean arterial pressure) occurred after 30 min. There was a linear relationship between the changes in mean arterial pressure and log plasma concentration of felodipine. Cardiac output (dye dilution) increased during maximal blood pressure reduction, from 5.3 +/- 1.0 to 6.6 +/- 2.4 L/min (p less than 0.01), partly because of increased heart rate from 57 +/- 4 to 65 +/- 9.1 beats/min (p less than 0.01) and partly because of increased stroke volume from 93 +/- 14 to 104 +/- 33 ml (p less than 0.05). Renal plasma flow (para-aminohippuric acid clearance) increased significantly (p less than 0.05) from 343 +/- 138 to 391 +/- 154 ml/min, while glomerular filtration rate ([51Cr]EDTA clearance) did not change. Arteriovenous noradrenaline difference increased 36% during felodipine therapy, when corrected for blood flow increase. We conclude that felodipine is a calcium inhibitor with potent vasodilating properties.

Effects of verapamil--a calcium inhibitor--on the vasomotor response of cortical and juxtamedullary glomeruli of the human kidney.

Cortical and juxtamedullary glomeruli of the human kidney were isolated by sieving. Verapamil, a calcium inhibitor, was added to a suspension of the glomeruli. The vasomotor response was measured by a video analyser. We were thus able to determine the modification of the cortical and juxtamedullary glomeruli surface area. Only the cortical glomeruli became vasodilated in response to the calcium inhibitor.

Renal side effect of nifedipine.

The possibility of a renal-related side effect from nifedipine has been investigated by comparing the effects on plasma creatinine level of coronary arteriography in two groups of patients: one group treated with nifedipine and the other treated with various drugs but without calcium inhibitor. Results showed a moderate (mean, 13%) but highly significant rise in the 27 patients receiving various drugs but no calcium inhibitor. Plasma creatinine change was not significant (mean, 3%) in the 26 patients treated with nifedipine. In the conditions of this study, this difference could only be related to nifedipine treatment. In conclusion, nifedipine may have exerted a protective effect against radiocontrast media nephrotoxicity, which deserves further investigation.

Peripheral large arteries and the response to antihypertensive treatment.

Since systolic pressure is governed by the rate of ventricular ejection and the rigidity of the aortic wall, antihypertensive agents may have different effects on systolic and diastolic pressure. Despite an adequate decrease in diastolic pressure, systolic pressure may remain elevated due to structural alterations of large arteries. In the present study, a procedure is described to distinguish the dilation of small and large arteries. The former is evaluated from the calculation of forearm resistance and the latter from the determination of the arterial diameter of the brachial artery, using a bidimensional pulsed Doppler system. Nitroglycerin dilates the brachial artery, with no change in forearm resistance. Dihydralazine reduces the diameter of the brachial artery but decreases forearm resistance. Only calcium and converting-enzyme inhibitors dilate both small and large arteries and cause an increase in brachial blood flow.

The effect of calcium and prostaglandin inhibitors on the intestinal fluid response to heat-stable enterotoxin of Escherichia coli.

The role of calcium and prostaglandins in the intestinal fluid response to Escherichia coli heat-stable enterotoxin (ST) was investigated in infant mice. Drugs that inhibit calcium uptake-cromolyn sodium, diltiazem, and nifedipine-caused a significant (P less than 0.02, P less than 0.02, and P less than 0.01, respectively) decrease in the fluid response when administered with ST. The effect of cromolyn sodium and nifedipine was dose-dependent; both agents were effective when administered 30 min before toxin challenge. Inhibitors of prostaglandin synthesis-quinacrine hydrochloride and zomepirac sodium-caused a significant (P less than 0.05 and P less than 0.02, respectively) reduction in the fluid response when administered with or 30 min before ST. The fluid response to cyclic 8-bromoguanosine 3',5'-monophosphate or to nitroprusside, which directly activated guanylate cyclase, was not altered by inhibitors of calcium uptake or prostaglandin synthesis. These observations indicate that calcium and prostaglandins are involved in the initial events that result in an intestinal fluid response to E. coli ST.