Inhibition of tumor cell-platelet interactions and tumor metastasis by the calcium channel blocker,nimodipine

Nimodipine, a dihydropyridine calcium channel blocker, was evaluated in vitro for its ability to inhibit platelet aggregation induced by B 16 amelanotic melanoma (B16a) and Walker 256 carcinosarcoma (W256) cells, and for its ability to inhibit platelet-enhanced B16a and W256 adhesion to rat microvascular endothelial cells. Nimodipine produced a dose-dependent inhibition of tumor-cell-induced platelet aggregation (TCIPA). Platelets enhanced tumor cell adhesion to endothelium both in the presence and absence of overt platelet aggregation. However, the greatest enhancement of adhesion occurred under aggregatory conditions. Nimodipine at a dose of 40 µg/ml inhibited platelet-enhanced adhesion to endothelium under aggregatory and nonaggregatory conditions. Nimodipine was tested in vivo for its ability to inhibit both experimental and spontaneous metastasis. Nimodipine produced a 46 per cent inhibition of lung colony formation at a dose of 5 mg/kg body-weight. Over a dose range of 0·1–80 mg/kg, nimodipine produced a significant dose-dependent inhibition in the formation of lung metastases from a subcutaneous tumor. The in vitro results demonstrate that a dihydropyridine calcium channel blocker can inhibit tumor cell-platelet-endothelial cell interactions. The in vivo results suggest that these compounds may be a new class of antimetastatic agent.     

Thickening of the adrenal zona glomerulosa in dogs induced by oxodipine, a calcium channel blocker.

Subchronic effects of oxodipine, a calcium channel blocker affecting the adrenal gland of the dog, are described. Thirteen wk of treatment at a high dose (24 mg/kg/day) of oxodipine resulted in drug-induced thickening of the zona glomerulosa and increased stimulation of its secretory activity. It is postulated that subchronic administration of oxodipine at this dosage resulted in a decrease in blood pressure, with uninterrupted stimulation of the adrenal zona glomerulosa to release aldosterone, causing an increase in the width of this portion of the gland involving cellular hyperplasia. Support for this indirect effect is found in the increased presence of renin granules in the juxtaglomerular apparatus.     

Suppression of rat carotid lesion development by the calcium channel blocker PN 200-110.

Balloon catheter damage of the rat carotid artery endothelium results in an extensive and reproducible neointimal lesion composed of smooth muscle cells and connective matrix. The authors have examined two calcium channel blockers, PN 200-110 and PY 108-068, for their ability to inhibit neointimal lesion development in the rat carotid model. When given subcutaneously (1.0 mg/kg day) both compounds produced rapidly acting and long-lasting hypotension, reducing blood pressure 25-29%. At this dose given daily, PN 200-110 reduced lesion cross-sectional area by 44%, compared with only 25% seen by PY 108-068, which suggests that the antiatherosclerotic effect may not be related to lowering of blood pressure. Furthermore, PN 200-110 did not reduce the extent of platelet deposition (compared with controls) occurring at the denuded vessel surface 1 hour or 24 hours after balloon catheterization, which indicates that the inhibition of lesion development may not reflect an antiplatelet mechanism. The observed inhibition by PN 200-110 may relate to mitogen responses of the smooth muscle cell in the vessel wall (migration and proliferation) involved in lesion progression after endothelial damage.     

Prevention of diabetic vascular calcification by nifedipine, a dihydropyridine-based calcium channel blocker

Vascular calcification is a common feature in advanced atherosclerosis and also a predictor of future cardiovascular events such as unstable angina and myocardial infarction, especially in diabetes. There is a growing body of evidence that advanced glycation end products (AGEs), senescent macroprotein derivatives formed at an accelerated rate in diabetes, exist within atherosclerotic lesions, thereby being implicated in the pathogenesis of accelerated atherosclerosis in diabetes. Indeed, we have previously shown that AGE - their receptor (RAGE) interaction could induce angiogenesis through autocrine production of vascular endothelial growth factor, suggesting its role for plaque formation and enlargement in diabetes. Furthermore, we have found that AGEs have the ability to induce the osteoblatic differentiation of pericytes, thus contributing to the development of vascular calcification as well. These observations suggest that the inhibition of AGE formation or blockade of the downstream signaling of RAGE may be a novel therapeutic target for the inhibition of vascular calcification in diabetic atherosclerosis. Since we, along with others, have shown that nifedipine inhibits glycation of low-density lipoprotein in vitro and blocks the AGE-induced RAGE expression in endothelial cells through its anti-oxidative properties, nifedipine could inhibit vascular calcification by blocking the AGE formation or the downstream signaling in diabetes. In this paper, we would like to propose the possible ways of testing our hypothesis. Does nifedipine treatment slow down the progression of coronary calcification in diabetic patients? If the answer is yes, is this beneficial effect of nifedipine superior to that of other DHPs with equihypotensive properties? Does nifedipine treatment decrease expression levels of AGEs and RAGE in diabetic atherosclerosis? Is the unique effect of nifedipine on vascular calcification correlated with its AGE or RAGE-suppressing properties? These prospective studies will provide further valuable information whether nifedipine could prevent vascular calcification in diabetic atherosclerosis by blockade the AGE-RAGE signaling in vascular wall cells.     

Antiepileptic effects of IOS-1.1212, a new calcium channel blocker

Research Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Institute of Organic Synthesis, Latvian Academy of Sciences, Riga. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 112, No. 10, pp. 362–365, October, 1991.     

Antireproductive effect of the calcium channel blocker amlodipine in male rats.

The purpose of the present study was to investigate whether treatment of male rats with the calcium antagonist amlodipine, used in the treatment of hypertension and angina, interferes with the reproductive function of male rats. Amlodipine treatment (0.04 mg amlodipine besylate/rat/day for 30 days) decreased plasma follicle-stimulating hormone and testosterone but not luteinizing hormone or prolactin concentrations (measured by double-antibody radioimmuno-assay). A significant reduction (23%) was observed in sperm density (sperm suspension collected from the cauda epididymidis) as well as in the amount of mature spermatids (14%) and Sertoli cells (9%) counted in seminiferous tubule cross-sections (400 x magnification). The results reveal the deleterious effects of subacute amlodipine treatment on the reproductive function of male rats.     

Pregnancy following discontinuation of a calcium channel blocker in the male partner

The fertility potential of human sperm populations can be assessedby the presence of head-directed mannose ligand receptors (mannose-specificlectin) and the occurrence of spontaneous acrosome reactionsafter incubation under capacitating conditions in vitro. Wehave reported previously on the interaction between anti-hypertensivemedications and their effects on these parameters of male fertilitypotential. In this report we document the effects of cessationof calcium ion channel blocker medication on male fertility.Motile spermatozoa from a 30 year old infertile patient on acalcium ion channel blocker as anti-hypertensive treatment hadsubnormal expression of mannose-specific lectin and did notexhibit spontaneous acrosome reactions. Three months followingdiscontinuation of the medications, complete recovery of boththe expression of head-directed mannose ligand receptors andthe acrosome reaction was documented, though sperm motilityand morphology remained unchanged. The couple had 2 years ofinfertility and previously failed to conceive through sevencycles of Pergonal/intra-uterine insemination. Conception occurredon the second Pergonal/intra-uterine insemination cycle afterthe husband discontinued calcium ion channel blocker medication.Calcium ion channel blockers may adversely affect sperm fertilizingpotential. Discontinuation of such medications enhances thechances for conception.     

Suppression of epileptiform burst discharges in CA3 neurons of rat hippocampal slices by the organic calcium channel blocker,verapamil

Summary We studied the effects of the organic calcium channel blocker, verapamil, on spontaneous and bicuculline-induced epileptiform burst discharges in CA3 pyramidal cells of hippocampal slices. A transient increase of burst discharge rate was observed in most cells within 30 min after the addition of verapamil (100 M) to the perfusing medium. Prolonged verapamil perfusions gradually reduced the rate and duration of burst discharges, then abolished them in all tested slices (over periods of 50–150 min) without blocking synaptic transmission. Responses to intracellular injections of current pulses were also gradually affected by verapamil: Action potential amplitude was decreased, action potential duration increased, frequency adaptation increased, amplitude of the fast hyperpolarization following a single action potential decreased, and amplitude and duration of the slow afterhyperpolarization markedly reduced. The amplitude of calcium spikes elicited in slices perfused with tetrodotoxin-containing medium was not affected by verapamil, but the mean velocity of depolarization near the peak of the calcium spike was decreased. Membrane resting potential and input resistance were not affected by verapamil. These results confirm that verapamil is able to suppress epileptiform activity, but suggest that this effect is rather non-specific, due to inhibition of both postsynaptic sodium and calcium conductances.     

ω-Phonetoxin-IIA: a calcium channel blocker from the spider Phoneutria nigriventer

 A peptide with neurotoxic effect on mammals, purified from the venom of the spider Phoneutria nigriventer, was studied regarding its primary structure and its effects on voltage-gated calcium channels. The peptide, named ω-phonetoxin-IIA, has 76 amino acids residues, with 14 Cys forming 7 disulphide bonds, and a molecular weight of 8362.7 Da. The neurotoxicity is a consequence of the peptide’s blocking effects on high-voltage-activated (HVA) calcium channels. N-type HVA calcium channels of rat dorsal root ganglion neurons are blocked with affinity in the sub-nanomolar concentration range. The toxin also blocks L-type channels of rat β pancreatic cells, with an affinity 40 times lower. Although not studied in detail, evidence indicates that the toxin also blocks other types of HVA calcium channels, such as P and Q. No effect was observed on low-voltage-activated, T-type calcium channels. The significant homologies between ω-phonetoxin-IIA and the peptides of the ω-agatoxin-III family, and the overlapping inhibitory effects on calcium channels are discussed in terms of the structure-activity relationship. Received: 2 February 1998 / Received after revision and accepted: 23 March 1998     

Modulation of inward rectifier potassium channel by toosendanin, a presynaptic blocker.

The effect of toosendanin, a presynaptic blocker, on the inward rectifier potassium channel (K(Kir)) of hippocampal CA1 pyramidal neurons of rats was studied by the single-channel patch-clamp technique. The results showed that toosendanin had an inhibitory effect on K(Kir) in an excised inside-out patch of the neuron under a symmetrical 150 mM K(+) condition. By decreasing the slower open time constant and increasing the slower close time constant, toosendanin (1x10(-6)-1x10(-4) g/ml) significantly reduced the open probability of the channel in a concentration-dependent manner. Meanwhile, a dose-dependent reduction in unitary conductance of the channel was also detected after toosendanin application. These data offer an explanation for toosendanin-induced facilitation of neurotransmitter release and antibotulismic effect of the drug.     

Protective effect of diltiazem (a calcium channel blocker) against cadmium-induced toxicity in mice.

Protective efficacy of diltiazem (a calcium channel blocker) has been studied against cadmium chloride (CdCl2) induced hematological and biochemical alterations in Swiss albino mice. CdCl2 (5 mg/kg b.wt.; i.p.) with or without prior treatment of diltiazem (100 mg/kg b. wt.; i.p.) was given to six-week old mice. Significant increase in the number of bone marrow cells as well as hematological parameters was observed in diltiazem pretreated CdCl2 intoxicated animals. A significant increase in lipid peroxidation (LPO) and acid phosphatase (ACP) level, and decrease in glutathione (GSH) and alkaline phosphatase (ALP) level in blood as well as liver were measured in CdCl2 intoxicated mice, while such values were near normal in DTZ pretreated animals. Furthermore, a significant increase in erythropoeitin (EPO) level was observed in diltiazem (DTZ) pretreated CdCl2 intoxicated animals as compared to CdCl2 alone treated animals. Thus, Diltiazem administration before cadmium intoxication protects bone marrow and hematological constituents in mice.     

The effect of inhaled gallopamil, a potent calcium channel blocker, on the late-phase response in subjects with allergic asthma.

The effect of gallopamil on the late-phase response to inhaled allergen was evaluated in six young adults with allergic asthma in a crossover manner. During 2 study days, subjects received 20 mg of inhaled gallopamil or placebo 30 minutes before challenge with the same dose of allergen. In addition, a histamine challenge was performed 1 1/2 hours before and 24 hours after allergen challenge. On 2 additional study days, in the absence of allergen, basal airway responsiveness to histamine was measured before and after gallopamil or placebo administration. During the early phase, the mean +/- SD decrease in FEV1 was 28.0% +/- 11.3% after placebo and 25.1% +/- 8.4% after gallopamil administration (p greater than 0.05; beta = 0.14). During the late phase, the maximum decrease in FEV1 was 26.9% +/- 11.9% after placebo and 25.3% +/- 10.3% after gallopamil administration (p greater than 0.05; beta = 0.21). Airway reactivity to histamine 24 hours after allergen challenge could not be measured in three subjects after gallopamil administration and in one subject after placebo administration because of persistent bronchospasm. In contrast, basal responsiveness to histamine in the absence of allergen was modestly decreased by gallopamil. Since gallopamil is one of the most potent calcium channel blockers when it is administered by the inhaled route, it is unlikely that this group of drugs will be clinically useful for allergic asthma.     

Regulation of the rat sarcoplasmic reticulum calcium release channel by calcium

The regulation by calcium of the ryanodine receptor/SR calcium release channel (RyR) from rat skeletal muscle was studied under isolated conditions and in situ. RyRs were either solubilized and incorporated into lipid bilayers or single fibres were mounted into a Vaseline gap voltage clamp. Single channel data were compared to parameters determined from the calculated calcium release flux. With K⁺ (250 mM) being the charge carrier the single channel conductance was 529 pS at 50 M Ca²⁺ cis and trans, and decreased with increasing cis [Ca²⁺]. Open probability showed a bell shaped calcium dependence revealing an activatory and an inhibitory Ca²⁺ binding site (Hill coefficients of 1.18 and 1.28, respectively) with half activatory and inhibitory concentrations of 9.4 and 298 M. The parameters of the inhibitory site agreed with the calcium dependence of channel inactivation deduced from the decline in SR calcium release in isolated fibres. Mean open time showed slight [Ca²⁺] dependence following a single exponential at every Ca²⁺ concentration tested. Closed time histograms, at high [Ca²⁺], were fitted with three exponentials, from which the longest was calcium independent, and resembled the recovery time constant of SR inactivation (115 ± 15 ms) obtained in isolated fibres. The data are in agreement with a model where calcium binding to the inhibitory site on RyR would be responsible for the calcium dependent inactivation in situ.     

Inhibition of excitation-contraction coupling by a Ca channel blocker nicardipine at low temperature in frog twitch fibers

Effects of a Ca channel blocker, nicardipine, on excitation-contraction (E-C) coupling were investigated in single twitch fibers dissected from short skeletal muscles of the frog, Rana japonica. The treatment with 20 microM nicardipine at 25 degrees C evoked a reversible twitch potentiation. By contrast, the treatment at the same concentration at 5 degrees C for 30 min evoked an intense twitch inhibition. The inhibition was irreversible. This paralyzed fiber remained at nearly normal resting and propagated action potentials. The treatment with 30 microM nicardipine for 14 min at 6 degrees C completely inhibited a potassium contracture by 190 mEq/l potassium ion for 1.5 h after the removal of nicardipine despite the retention of normal resting potentials. The paralyzed fiber which was previously treated with 20 microM nicardipine for 30 min at 6 degrees C, also remained at normal action potential and at partial potassium contracture, and responded to 3 mM caffeine stimulation with a normal contracture. Dose-dependence curves obtained from the data on the treatments with nicardipine at various concentrations for 30 min at 6 degrees C consisted of an opposite sigmoidal shape with both threshold and half maximum inhibition at 1 and 1.5 microM for twitch and similarly at 5 and 12 microM for potassium contracture. Temperature-dependence curves obtained from the data on the treatments with 20 microM nicardipine for 30 min at various temperatures consisted of a sigmoidal shape with half maximum inhibition at 15 degrees C for twitch and at 12 degrees C for potassium contracture. A working hypothesis "two component-three state model" for E-C coupling was proposed based on the interpreted mechanism of nicardipine action.     

RWJ-22108 — a novel airway tissue — selective calcium channel blocker

RWJ-22108 is a novel calcium entry blocker that has potential therapeutic use as an antiasthmatic agent. Although displaying typical potent inhibition of⁴⁵Ca uptake into aortic rings (IC₅₀=7.1 nM) and displacement of [³H]nitrendipine from cardiac membranes (IC₅₀=137 nM), RWJ-22108 demonstrates tissue selectivity in the inhibition of KCl-induced contractions. RWJ-22108 inhibits the calcium-dependent contraction of canine bronchiolar smooth muscle with an IC₅₀ of 5.7 nM. The IC₅₀ femoral artery/IC₅₀ bronchiolar ratios are 2.85, 8.02, 1.47 and 1.96 for nifedipine, RWJ-22108, verapamil, and gallopamil, respectively. Furthermore, this selectivity ratio (range 2.8–5.5) of RWJ-22108 is also observed when inhibition of other pulmonary and cardiovascular smooth muscles are compared. Using canine tracheal muscle and rabbit aortae, the IC₅₀ aorta/IC₅₀ trachea ratio is 1.75 for RWJ-22108 compared to approximately 0.5 for several calcium blocker standards. These results indicate thatin vitro RWJ-22108 is a bronchoselective calcium channel blocker.     

Old age and cognition: enhancement of recent memory in aged rats by the calcium channel blocker nimodipine.

Aged hooded rats were given extensive training on an 8-arm radial arm maze task. The testing procedure was so designed as to maximally challenge each animal's trial specific memory capacities. After performance had stabilized, the animals were administered different dosages of the dihydropyridine calcium channel blocker nimodipine. In all animals, the drug improved performance in the 8-arm maze but, more importantly, it did so in a dose-related manner. These results, along with other findings, support the notion that calcium homeostasis may be an important factor determining the behavioral consequences of aging and particularly memory dysfunctions.     

Hippocampal synaptic plasticity induced by excitatory amino acids includes changes in sensitivity to the calcium channel blocker, omega-conotoxin

The hippocampus is widely used in investigations of different forms of synaptic plasticity, including long-term potentiation and kindling. Receptors for excitatory amino acids (EAAs) play a prominent role in these phenomena. Recently, is has been demonstrated that exposure of hippocampal slices to EAAs and related agonists produces biphasic effects on excitatory synaptic transmission: initial blockade of synaptic responses is followed by a delayed recovery. The recovered responses demonstrate altered pharmacological properties: they acquire sensitivity to N-methyl-D-aspartate (NMDA) antagonists during L-glutamate (Glu) exposure and lose sensitivity to both NMDA and non-NMDA antagonists under L-aspartate (Asp). These changes persist for many hours. It was suggested that this form of hippocampal plasticity may involve transitions between distinct states of synaptic functioning. To explore this possibility, we investigated several properties of synaptic transmission in the initial and EAA-modified states. Here we report that hippocampal postsynaptic potentials (PSPs) evoked under Glu or Asp exposure completely lose sensitivity to omega-conotoxin GVIA (omega-CgTX), a potent, specific, and irreversible blocker of certain types of neuronal calcium channels. After washout of the EAA, sensitivity to the toxin is regained. These results indicate that prolonged EAA exposure induces profound changes in the machinery of synaptic transmission, which include, but are not limited to, changes in calcium channel functioning.     

Revival of nifedipine, a dihydropyridine-based calcium blocker

A recent analysis by the Blood Pressure Lowering Treatment Trialists' Collaboration revealed that any commonly-used blood pressure (BP)-lowering regimen reduced the risk of total major cardiovascular events, and larger reductions in BP produced larger reductions in the risk. These observations suggest that most of the differences among treatment regimens in their effects on cardiovascular outcomes could be explained by the differences in achieved BP level. However, it may also be true that some treatment regimen is superior or inferior to others with regard to the risk reduction of cardiovascular events. Indeed, the data from dihydropyridine-based calcium antagonist (DHP) trails are consistent in that they could not protect against new-onset heart failure or progression of renal disease in patients with left-ventricular systolic dysfunction or overt proteinuria, respectively. However, a multicenter, randomized, placebo-controlled, double-blind ACTION trial, which compared the effect of long-acting nifedipine or placebo on mortality and cardiovascular morbidity in patients with stable angina, revealed that nifedipine reduced the risk for new-onset overt heart failure by 29%. Further, an open-label, randomized prospective J-MIND trial, which compared the effect of long-acting nifedipine or enalapril, an angiotensin-converting enzyme inhibitor on onset and progression of nephropathy in hypertensive patients with type 2 diabetes, showed that long-acting nifedipine had an equipotent reoprotective effect on diabetic nephropathy. In this paper, we would like to propose our hypothesis that nifedipine may be unique and superior in its effects on heart failure and proteinuria compared with various DHPs. For ensuring our hypothesis, the following clinical issues would be addressed. Does nifedipine treatment alone decrease the progression of renal disease with overt proteinuria? If these answers are yes, are these beneficial effects of nifedipine superior to that of other DHPs with equihypotensive properties? Does nifedipine treatment also reduce oxidative stress markers? Are these unique effects of nifedipine correlated with its anti-oxidative properties? These prospective studies will provide further valuable information whether nifedipine may be a preferred DHP to achieve BP goals in hypertensive patients with systolic dysfunction or overt proteinuria.     

Efficacy of MEM 1003, a novel calcium channel blocker, in delay and trace eyeblink conditioning in older rabbits

Eyeblink conditioning is a relatively simple form of associative learning that shows neurobiological and behavioral parallels across several species, including humans. Aged subjects acquire eyeblink conditioning more slowly than young ones. In addition, eyeblink conditioning effectively discriminates patients with Alzheimer's disease from healthy older adults. The present study evaluated the effect of a novel L-type Ca2+ channel antagonist, MEM 1003, on delay and trace eyeblink conditioning in older (mean 33.4 months old) female New Zealand white rabbits. In the delay conditioning paradigm, an 850 ms tone conditioning stimulus (CS) was followed 750 ms after its onset by a 100 ms corneal air puff. Several trace conditioning paradigms were evaluated, with a silent period of 300, 400 or 500 ms between the end of the tone CS and the delivery of the air puff. Learning was more difficult in the longer trace paradigms than in the delay paradigm. MEM 1003, at a dose of 2.0 mg/kg, s.c., given daily 30 min prior to training on each of the 15 training days, enhanced learning compared to vehicle injections in both delay and trace paradigms. However, higher or lower doses were ineffective. These results support previous work demonstrating that modulation of Ca2+ channel activity can reduce age-related cognitive impairments.