Effect of verapamil on ventilation and chemical control of breathing in anesthetized rats

The calcium channel blocker, verapamil (0.1-1.0 mg/kg, i.v.) was administered to anesthetized rats to determine its effects on ventilation and on ventilatory responses to hypoxia and CO2. Verapamil produced a dose-dependent increase in tidal volume (VT) and a decrease in respiration rate (f). The bradypnea due to verapamil was characterized by an increase in expiratory duration (TE) and no change of inspiratory duration (TI). Verapamil produced similar changes in VT and f in vagotomized rats. The increase in respiration rate and minute volume due to hypoxia were inhibited by verapamil (0.5 and 1.0 mg/kg) but the increase in tidal volume due to hypoxia was depressed only with the 1.0 mg/kg dose. On the other hand, the increase in VT due to breathing CO2 was not changed by verapamil (0.1-1.0 mg/kg), but depression of the respiratory frequency response to CO2 occurred with 1.0 mg/kg of verapamil. These results indicate that verapamil produced slow, deep breathing and these responses were not mediated by vagal mechanisms. Ventilatory responses to hypoxia were depressed by verapamil. However, since the calcium blocker demonstrated no effect on the VT-CO2 relationship, verapamil did not change ventilatory chemosensitivity to CO2. The data also suggest that mechanisms governing the control of respiratory frequency are more sensitive to verapamil than tidal volume responses.     

Acrylonitrile: in vivo cytogenetic studies in mice and rats

Acrylonitrile (VCN), a suspect human carcinogen, does not produce significant increases in cytogenetic aberrations in the mouse-bone marrow when given orally for 4, 15 or 30 days at doses equal to 7, 14 and 21 mg/kg/day resp. or by i.p. for the same time periods at doses of 10, 15 and 20 mg/kg/day. Rats treated orally with 16 daily doses of VCN (40 mg/kg/day) or potassium cyanide (KCN) (5 mg/kg/day) showed no increase of aberrant metaphases in the bone marrow over controls.     

Pre-electroconvulsive shock administration of calcium channel blockers reduces retrograde amnesia induced by ECS

Effect of pre-electroconvulsive shock (ECS) administration of calcium channel blockers (CCBs) like verapamil, diltiazem, nifedipine, nimodipine, flunarizine and cinnarizine on retrograde amnesia induced by ECS was examined using passive avoidance paradigm in rats. The groups (Gr 1-7) of adult, male Wistar rats received true ECS with CCBs (5mg/kg; i.p) or vehicle (10 ml/kg; ip) and other groups (Gr 8-14) received sham ECS with CCBs (5mg/kg; i.p) or vehicle (10 ml/kg; i.p). The anti-amnestic activity of CCBs were evaluated using the passive avoidance paradigm in rats. Results showed that, the baseline latencies for all the groups did not differ significantly. Rats receiving true ECS produced significantly lower latencies. There was increase in the post ECS step through latencies of the rats administered CCBs before ECS. Therefore, pre-ECS administration of calcium channel blockers might reduce retrograde amnesia produced by ECS without altering seizure duration.     

Effects of the calcium channel blocker flunarizine on the hemodynamics and oxygenation of tumor microvasculature.

Flunarizine is a diphenylpiperazine calcium entry blocker that has been shown previously to increase tumor blood flow and sensitivity to radiotherapy via reduction in the radiobiologically significant hypoxic fraction. Two mechanisms of action have been proposed previously (vasodilation, altered blood viscosity), but no studies have been performed to examine its mechanisms of action in vivo. Such information would be invaluable in determining the role of flunarizine in multimodality approaches to reduce tumor hypoxia. Fisher-344 rats bearing R3230Ac tumors transplanted into dorsal flap window chambers were used to examine microcirculatory changes after administration of flunarizine (1.0 mg/kg, iv). The drug increased the diameters of the microvasculature and red cell velocities specifically in central tumor regions (producing an average increase in vessel flow by a factor of 1.96), which was accompanied by an increase in perivascular pO2 of 12 mm Hg, on the average. The drug did not change the diameters of tumor "feeding" vessels, nor did it change vascular length densities. Thus the improvement in central tumor blood flow and oxygenation could not be attributed to dilation of feeding vessels. The oxygen-carrying capacity of the blood was not altered either since hemoglobin saturation (measured in vitro) and the hematocrits of the microvasculature were unchanged after drug administration. Therefore, by a process of elimination, the most likely explanation for the effect of the drug is modification of blood viscosity. Additional studies are under way in this laboratory to examine whether changes in viscosity occur after flunarizine administration.     

Augmented behavioral response and enhanced synaptosomal calcium transport induced by repeated cocaine administration are decreased by calcium channel blockers

Recent studies suggest that calcium influx via L-type calcium channels is necessary for psychostimulant-induced behavioral sensitization. In addition, chronic amphetamine upregulates subtype Cav1.2-containing L-type calcium channels. In the present studies, we assessed the effect of calcium channel blockers (CCBs) on cocaine-induced behavioral sensitization and determined whether the functional activity of L-type calcium channels is altered after repeated cocaine administration. Rats were administered daily intraperitoneal injections of either flunarizine (40 mg/kg), diltiazem (40 mg/kg) or cocaine (20 mg/kg) and the combination of the CCBs and cocaine for 30 days. Motor activities were monitored on Day 1, and every 6th day during the 30-day treatment period. Daily cocaine administration produced increased locomotor activity. Maximal augmentation of behavioral response to repeated cocaine administration was observed on Day 18. Flunarizine pretreatment abolished the augmented behavioral response to repeated cocaine administration while diltiazem was less effective. Measurement of tissue monoamine levels on Day 18 revealed cocaine-induced increases in DA and 5-HT in the nucleus accumbens. By contrast to behavioral response, diltiazem was more effective in attenuating increases in monoamine levels than flunarizine. Cocaine administration for 18 days produced increases in calcium uptake in synaptosomes prepared from the nucleus accumbens and frontal cortex. Increases in calcium uptake were abolished by flunarizine and diltiazem pretreatment. Taken together, the augmented cocaine-induced behavioral response on Day 18 may be due to increased calcium uptake in the nucleus accumbens leading to increased dopamine (DA) and serotonin (5-HT) release. Flunarizine and diltiazem attenuated the behavioral response by decreasing calcium uptake and decreasing neurochemical release.     

The effects of indomethacin and verapamil on dextran-induced shock in rats

To clarify the mechanism of the anti-shock effect of indomethacin, dextran-induced shock in rats was used as a shock model and compared with the effect of verapamil, a calcium antagonist. Thirty minutes after pretreatment with indomethacin or verapamil, 5% dextran (1 ml/kg body weight) was iv. infused into rats. Pretreatment with indomethacin (5 mg/kg iv.) or verapamil (2 mg/kg iv.) 30 min prior to the dextran infusion prevented significantly a decrease in blood and pulse pressure, and also an increase in the hematocrit value, paw thickness and serum histamine level of rats. Neither pretreatment with indomethacin (or verapamil) nor saline control changed the serum prostaglandin E level before and after the dextran infusion. The effects of verapamil in preventing dextran-induced shock were found to be much greater than those of indomethacin. These results indicated that the shock-preventing effect of indomethacin may be ascribed, at least to some degree, to the role of the drug as a calcium antagonist like verapamil rather than as a cyclooxygenase inhibitor which lowers prostaglandins and/or thromboxane levels. The former effect may be exerted on the mast cells to inhibit calcium influx stimulated by dextran, resulting in the prevention of histamine release.     

Effects of acute and chronic nicotine on elevated plus maze in mice: involvement of calcium channels

The current experiments examined the anxiety-related effects of acute and repeated nicotine administration using the elevated plus maze test in mice. Nicotine (0.1 mg/kg s.c., 5 and 30 min after injection; 0.5 mg/kg, s.c., 5 min after injection) had an anxiogenic effect, shown by specific decreases in the percentage of time spent on the open arms and in the percentage of open arm entries. Tolerance developed to this anxiogenic action after 6 days of daily nicotine administration (0.1 mg/kg, s.c.). Five minutes after the seventh injection, an anxiolytic effect was observed, i.e., specific increases in the percentage of time spent on the open arms and in the percentage of open arm entries. L-type voltage-dependent calcium channel antagonists nimodipine (5 and 10 mg/kg, i.p.), flunarizine (5 and 10 mg/kg, i.p.), verapamil (5, 10, 20 mg/kg) and diltiazem (5, 10, 20 mg/kg, i.p.) were also injected prior to an acute low dose of nicotine or to each injection of chronic nicotine. Our results revealed that calcium channel blockers dose-dependently attenuated both an anxiogenic effect of nicotine as well as the development of tolerance to this effect. Our results suggest that neural calcium-dependent mechanisms are involved in the anxiety-related responses to acute and chronic nicotine injection that may ultimately lead to addiction and smoking relapse in human smokers.     

Effects of amlodipine, diltiazem, and verapamil on the anticonvulsant action of topiramate against maximal electroshock-induced seizures in mice

To assess the effect of 3 calcium channel antagonists (amlodipine, diltiazem, and verapamil) on the anticonvulsant action of topiramate (a new generation antiepileptic drug) in the mouse maximal electroshock seizure (MES) model. Amlodipine (20 mg/kg) significantly enhanced the anticonvulsant activity of topiramate in the MES test in mice, reducing its ED50 value from 54.83 to 33.10 mg/kg (p < 0.05). Similarly, diltiazem (5 and 10 mg/kg) markedly potentiated the antiseizure action of topiramate against MES, lowering its ED50 value from 54.83 to 32.48 mg/kg (p < 0.05) and 28.68 mg/kg (p < 0.01), respectively. In contrast, lower doses of amlodipine (5 and 10 mg/kg) and diltiazem (2.5 mg/kg) and all doses of verapamil (5, 10, and 20 mg/kg) had no significant impact on the antiseizure action of topiramate. Pharmacokinetic verification of the interaction of topiramate with amlodipine and diltiazem revealed that neither amlodipine nor diltiazem affected total brain topiramate concentration in experimental animals, and thus, the observed interactions were concluded to be pharmacodynamic in nature. The favorable combinations of topiramate with amlodipine or diltiazem deserve more attention from a clinical viewpoint because the enhanced antiseizure action of topiramate was not associated with any pharmacokinetic changes in total brain topiramate concentration.     

Effects of verapamil on behavioral and microcirculatory disorders in pain syndrome of spinal etiology

In the experiments on Wistar rats with pain syndrome of spinal origin (PSSO) caused by the generator of pathologically enhanced excitation (GPEE) in dorsal horns of the spinal cord lumbosacral segments, it was shown that the intravenous verapamil injection (1.25 mg/kg) undoubtedly decreased behaviour response and improves the state of microcirculation. The compound of 10-fold decreased dose does not affect the behaviour response and microcirculation. When PSSO exists, the intravenous injection of analgin (150 mg/kg) produced an effect on the behaviour response and does not produce any action on microcirculation. When verapamil reaches the dorsal surface of the spinal cord (GPEE area) it decreases the behaviour response and microcirculation disorders created in PSSO. The obtained data make it clear that the GPEE depression caused by the verapamil calcium channel blocker weakens PSSO and normalizes the microcirculation.     

Allicin protects against myocardial apoptosis and fibrosis in streptozotocin-induced diabetic rats

To evaluate the cardioprotective effect of allicin (AL) on myocardial injury of streptozotocin (STZ)-induced diabetic rats and to further explore its underlying mechanisms. Hyperglycemia was induced in rats by single intraperitoneal injection of STZ (40 mg/kg). Three days after STZ induction, the hyperglycemic rats (plasma glucose levels ≥ 16.7 mmol/l) were treated with AL by intraperitoneal injection at the doses of 4 mg/kg, 8 mg/kg, and 16 mg/kg daily for 28 days. The fasting blood glucose levels were measured on every 7th day during the 28 days of treatment. The body weight, blood glucose, and parameter of cardiac function were detected after 4 weeks to study the cardioprotective effects of AL on diabetic rats in vivo. The apoptotic index of cardiomyocytes was estimated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The expressions of Fas, Bcl-2, CTGF, and TGF-β(1) protein were studied by immunohistochemistry. Laser scanning confocal microscopy technique was utilized to observe the effects of AL on intracellular calcium concentration ([Ca(2+)](i)) in rat ventricular cardiomyocytes. AL at the doses of 4 mg/kg, 8 mg/kg, and 16 mg/kg significantly reduced blood glucose levels in a dose-dependent manner and increased body weight as well compared with the model group. Hemodynamic parameters including left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and maximum rate of left ventricular pressure rise and fall (+dp/dtmax and -dp/dtmax) were significantly restored back to normal levels in AL-treated (8 mg/kg and 16 mg/kg) rats compared with diabetic model rats. AL markedly inhibited cardiomyocyte apoptosis induced by diabetic cardiac injury. Further investigation revealed that this inhibitory effect on cell apoptosis was mediated by increasing anti-apoptotic protein Bcl-2 and decreasing pro-apoptotic protein Fas. Additional experiments demonstrated AL abrogated myocardial fibrosis by blocking the expressions of CTGF and TGF-β(1) protein. AL shows protective action on myocardial injury in diabetic rats. The possible mechanisms were involved in reducing blood glucose, correcting hemodynamic impairment, reducing Fas expression, activating Bcl-2 expression, decreasing intracellular calcium overload, inhibiting the expressions of TGF-β(1) and CTGF, and further improving cardiac function.     

Pharmacological Characterization of Morphine-Induced In Vivo Release of Cholecystokinin in Rat Dorsal Horn

Previous studies indicate that an increased release of cholecystokinin (CCK) in response to morphine administration may counteract opioid-induced analgesia at the spinal level. In the present study we used in vivo microdialysis to demonstrate that systemic administration of antinociceptive doses of morphine (1-5 mg/kg, s.c.) induces a dose-dependent and naloxone-reversible release of CCK-like immunoreactivity (CCK-LI) in the dorsal horn of the spinal cord. A similar response could also be observed following perfusion of the dialysis probe for 60 min with 100 microM but not with 1 microM morphine. The CCK-LI release induced by morphine (5 mg/kg, s.c.) was found to be calcium-dependent and tetrodotoxin-sensitive (1 microM in the perfusion medium). Topical application of either the L-type calcium channel blocker verapamil (50 microg) or the N-type calcium channel blocker omega-conotoxin GVIA (0.4 microg) onto the dorsal spinal cord completely prevented the CCK-LI release induced by morphine (5 mg/kg, s.c.). Our data indicate that activation of L- and N-type calcium channels is of importance for morphine-induced CCK release, even though the precise site of action of morphine in the dorsal horn remains unclear. The present findings also suggest a mechanism for the potentiation of opioid analgesia by L- and N-type calcium channel blocking agents.     

Possible involvement of GABA mechanisms in central cardiovascular and respiratory control. Studies on the interaction between diazepam, picrotoxin and clonidine

Various doses (0.1-1 mg/kg) of diazepam were given to chloralose anesthetized rats, with both systemic (i.p.) and central injections being tested. Arterial pressure, heart rate, respiration depth and frequency were recorded. Diazepam caused a dose-dependent decrease in the arterial pressure after systemic administration and also decreased it after central administration. However, only intraventricular but not intracisternal injections of diazepam were effective. The hypotensive effect of systemic diazepam was competitively counteracted by pretreatment with picrotoxin, a putative gamma-aminobutyric acid receptor blocking agent. The hypotensive effect of the centrally acting alpha-adrenoreceptor agonist clonidine was not influenced by picrotoxin pretreatment. The effect of diazepam on heart rate was inconsistent. Diazepam caused a reduction of respiratory frequency, which was not counteracted by picrotoxin pretreatment. It is concluded that central gabergic mechanisms are to some extent involved in the hypotensive effect of diazepam, probably at a supramedullary level. The hypotensive effect of a threshold dose of diazepam was blocked by a small dose of clonidine. Likewise, diazepam pretreatment could counteract the hypotension, bradycardia and respiratory frequency reduction caused by a threshold dose of clonidine. These results suggest that gabergic and/or other benzodiazepine-sensitive receptors may interact with alpha-adrenoreceptors in the control of central cardiovascular and respiratory mechanisms.     

Cyanide-induced changes in the levels of neurotransmitters in discrete brain regions of rats and their response to oral treatment with alpha-ketoglutarate

Cyanide is a potential suicidal, homicidal and chemical warfare agent. It produces histotoxic hypoxia following inhibition of cytochrome c oxidase, a terminal respiratory chain enzyme. The profound metabolic changes lead to neurotoxicity including alterations in the levels of neurotransmitters. The present study addressed the effect of acute exposure of lethal and sub-lethal doses of potassium cyanide (KCN; 0.75 or 2.0 LD50; po) on the levels of neurotransmitters in discrete brain regions of rats and its response to treatment with alpha-ketoglutarate (alpha-KG; 0.5 g/kg; po; -10 min) alone or with sodium thiosulphate (STS; 1.0 g/kg; ip; -15 min). KCN significantly decreased norepinephrine, dopamine and 5-hydroxytryptamine levels in different brain regions which were resolved by alpha-KG and/or STS. Corpus striatum and hippocampus were more sensitive as compared to cerebral cortex and hypothalamus. alpha-KG, a potential cyanide antidote alone or with STS showed neuroprotective effects against cyanide.     

Inhibitory Effect of Calmodulin Antagonists and Calcium Antagonists on Fertilization-Induced Cyanide-Insensitive Respiration in Sea Urchin Eggs

During initial several minutes after fertilization, sea urchin eggs exhibited high rate of respiration which was only slightly inhibited by cyanide. This cyanide-insensitive respiration was inhibited by calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalensulfonamide hydrochloride (W-7), N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and chlorpromazine, which were added within 1 min after insemination. The inhibitory effect of W-7 on cyanide-insensitive respiration was higher than that of W-5. Cyanide-sensitive respiration of fertilized eggs observed after this initial period was not inhibited by these compounds. Ca²⁺ influx in eggs just after fertilization was inhibited by calcium antagonists but was rather enhanced by calmodulin antagonists. Fertilization-induced stimulation of cyanide-insensitive respiration probably results from calmodulin-dependent reactions which are activated by Ca²⁺ influx.     

Effect of verapamil on the aldosterone-stimulating properties of metoclopramide: in vitro and in vivo studies

The role of calcium in the regulation of aldosterone secretion has been recently clarified. Angiotensin II and potassium stimulate aldosterone secretion through a calcium-entry dependent mechanism, while ACTH action is both calcium and cyclic AMP dependent. To establish whether also the so-called aldosterone dopaminergic regulatory system is calcium-dependent we have studied, in vitro and in vivo, the effect of verapamil, a calcium entry blocker agent, on the aldosterone-stimulating properties of the antidopaminergic drug, metoclopramide. In the rat adrenal cells perfusion system, verapamil blocked both angiotensin II and metoclopramide-stimulated aldosterone. This effect on metoclopramide action seems to be present also in vivo in normal subjects: in fact aldosterone response was slightly but significantly reduced after pretreatment with verapamil. In conclusion the results suggest that also the dopaminergic system could regulate aldosterone secretion through calcium-mediated mechanisms.     

Organ-distribution of the metabolite 2-aminothiazoline-4-carboxylic acid in a rat model following cyanide exposure

The reaction of cyanide (CN(-)) with cystine to produce 2-aminothiazoline-4-carboxylic acid (ATCA) is one of the independent detoxification pathways of cyanide in biological systems. In this report, in vivo production of ATCA and its distributions in plasma and organs were studied after a subcutaneous sublethal dose of 4?mg/kg body weight potassium cyanide (KCN) administration to rats. At this sublethal dose of KCN, ATCA concentration was not significantly increased in the plasma samples, however, it was found significantly increased in liver samples. These results suggested that ATCA might not be a good diagnostic biomarker in plasma for sublethal cyanide exposure; however, liver could serve as the right organ for the detection of ATCA in post-mortem examinations involving cyanide exposure in military, firefighting, industrial and forensic settings.     

Effects of systemically administered monoamine reuptake blocking agents on patterns of buspirone-induced analgesia in rats

We have recently shown the serotonin 5-HT1A receptor agonist buspirone to produce analgesia in several pain tests in rats. As a 5-HT1A agonist, buspirone may change serotonergic (5-HT) tone to alter the balance of central monoaminergic (MA) systems that function in analgesia. MA-reuptake blocking drugs have been shown to produce analgesia, both when given alone and in combination with a variety of other agents, presumably via their action upon MA neurochemistry. The present study was undertaken to examine the effect of systemic administration of the 5-HT-reuptake blocker amitriptyline (AMI: 10 mg/kg), NE-reuptake blocker desipramine (DMI: 10 mg/kg) or DA-reuptake blocker GBR-12909 (7.5 mg/kg) on patterns of analgesia produced by buspirone (1-5 mg/kg) in thermal and mechanical pain tests in rats. Neither reuptake blocking agents or buspirone, when administered alone or in combination, produced overt changes in motor activity at the doses tested. AMI alone was not analgesic in either thermal or mechanical pain tests. In both assays, AMI reduced the analgesic action of buspirone, with greater effects seen in the thermal test. When administered alone, DMI produced significant analgesia against thermal and mechanical pain. DMI significantly attenuated the analgesic action of all doses of buspirone in both pain tests. Alone, GBR-12909 did not affect nociception in thermal or mechanical tests. GBR-12909 decreased buspirone-induced analgesia at all doses in the thermal test, while having no effect on buspirone-induced analgesia against mechanical pain. These results demonstrate that facilitation of 5-HT, NE and DA function with reuptake blocking drugs did not enhance the analgesic action of buspirone. These data indicate against the adjuvant use of reuptake blocking compounds and buspirone as analgesics. Furthermore, such findings may suggest other possible non-MA substrates of buspirone-induced analgesia.     

Ca2+ modulators as antidotes to imipramine and neurotransmitter toxicity

Flunarizine and nimodipine, Ca2+ modulators which exert antagonist effects against catecholamines and serotonin and have specific action on the brain, were used as antidotes to imipramine toxicity in the rat. Imipramine, a tricyclic antidepressant, inhibits synaptic reuptake of catecholamines and serotonin. Flunarizine administered concurrently with imipramine increased survival time significantly (p less than 0.04). After a lethal dose of imipramine (85 mg/kg) 5 out of 5 animals treated with flunarizine (2.37 +/- 1.21 mg/kg in divided doses) and 4 out of 5 animals treated with nimodipine (0.36 +/- 0.11 mg/kg) survived. The acute toxicity of imipramine might be related, in part, to drug-induced alteration in turnover of excitatory neurotransmitters which will induce intracellular Ca2+ accumulation and damage to vital organs. These toxic effects of endogenously produced neuroamines may be antagonized by nimodipine or flunarizine.     

The conditioned place preference paradigm in rats: effect of bupropion

The effect of bupropion in the conditioned place preference paradigm in rats is described. In doses between 10 and 50 mg/kg i.p. bupropion increased the time rats spent in a shuttle box compartment conditioned to this compound. This effect of bupropion was not blocked by pretreatment of the animals with 0.1 mg/kg i.p. haloperidol or 50 mg/kg i.p. sulpiride. The same doses of the neuroleptics did, however, attenuate locomotor hyperactivity induced by bupropion. Bupropion thus seems to belong to the group of CNS stimulants whose effect in the conditioned place preference paradigm is not blocked by neuroleptics.     

Streptozotocin and alloxan produce alterations in rat brain monoamines independently of pancreatic beta cells destruction.

In recent years the effect of experimental diabetes mellitus on brain neurochemistry has been under an intensive investigation. In most of these studies diabetes was produced by a peripheral administration of streptozotocin or alloxan. In line with previous reports, a week after such an application of alloxan (200 mg/kg s.c.) we found the concentration of serotonin, dopamine and norepinephrine to be increased in the brain of a diabetic rat. Accumulation of these monoamines, produced by inhibition of monoamine oxydase with pargyline (100 mg/kg i.p.) decreased in animals made diabetic by alloxan or streptozotocin (100 mg/kg i.p.) suggesting a decrease in deamination rate. Surprisingly, however, one week after an intracerebroventricular administration of non-diabetogenic doses of streptozotocin (5-20 mg/kg) or alloxan (20 mg/kg), changes in brain monoamines were similar to those observed in diabetic animals. This observation apparently suggests that the CNS effect of streptozotocin or alloxan is not necessarily related to a diabetogenic, beta-cytotoxic action of these substances.