Calcicludine binding to the outer pore of L-type calcium channels is allosterically coupled to dihydropyridine binding.

How dihydropyridines modulate L-type voltage-gated Ca2+ channels is not known. Dihydropyridines bind cooperatively with Ca2+ binding to the selectivity filter, suggesting that they alter channel activity by promoting structural rearrangements in the pore. We used radioligand binding and patch-clamp electrophysiology to demonstrate that calcicludine, a toxin from the venom of the green mamba snake, binds in the outer vestibule of the pore and, like Ca2+, is a positive modulator of dihydropyridine binding. Data were fit using an allosteric scheme where dissociation constants for dihydropyridine and calcicludine binding, KDHP and KCaC, are linked via the coupling factor, alpha. Nine acidic amino acids located within the S5-Pore-helix segment of repeat III were sequentially changed to alanine in groups of three, resulting in the mutant channels, Mut-A, Mut-B, and Mut-C. Mut-A, whose substitutions are proximal to IIIS5, exhibits a 4.5-fold reduction in dihydropyridine binding and is insensitive to calcicludine binding. Block of Mut-A currents by calcicludine is indistinguishable from wild-type, indicating that KCaC is unchanged and that the coupling between dihydropyridine and calcicludine binding (i.e., alpha) is disrupted. Mut-B and Mut-C possess KDHP values that resemble that of the wild type. Mut-C, the most C-terminal of the mutant channels, is insensitive to calcicludine binding and block. KCaC values for the Mut-C single mutants, E1122A, D1127A, and D1129A, increase from 0.3 (wild type) to 1.14, 2.00, and 20.5 microM, respectively. Together, these findings suggest that dihydropyridine antagonist and calcicludine binding to L-type Ca2+ channels promote similar structural changes in the pore that stabilize the channel in a nonconducting, blocked state.

A third-generation, long-acting, dihydropyridine calcium antagonist, azelnidipine, attenuates stent-associated neointimal formation in non-human primates.

BACKGROUND: Calcium antagonists have been shown to reduce atherogenesis and improve clinical outcomes in atherosclerotic vascular disease. No study has so far, however, addressed the effects of calcium antagonists on stent-associated neointimal formation. We therefore investigated whether a third-generation calcium antagonist, azelnidipine, attenuates in-stent neointimal formation in non-human primates. METHOD: Male cynomolgus monkeys were fed a high cholesterol diet for 4 weeks, and were randomly assigned to three groups: a vehicle group and two other groups treated with azelnidipine at 3 and 10 mg/kg per day for an additional 24 weeks (n = 12 each). Multi-link stents were then implanted in the iliac artery. RESULTS: Azelnidipine at the high dose reduced neointimal thickness (0.25 +/- 0.02 versus 0.19 +/- 0.02 mm; P < 0.05). Azelnidipine also reduced local oxidative stress and monocyte chemoattractant protein 1 (MCP-1) expression. No difference was found between the three groups in the degrees of injury score, inflammation score, plaque neovascularization, or plasma lipid levels. Azelnidipine also reduced MCP-1-induced proliferation/migration of vascular smooth muscle cells in vitro. CONCLUSIONS: This study demonstrated for the first time that azelnidipine attenuates in-stent neointimal formation associated with the reduced expression of MCP-1 and smooth muscle proliferation/migration in the neointima. These data in non-human primates suggest potential clinical benefits of azelnidipine as a 'vasculoprotective calcium antagonist' in patients undergoing vascular interventions.

Nuevas dihidropiridinas sintéticas con propiedades antagonistas del calcio vasoselectivas / New synthetic dihydropyridines with vasoselective calcium antagonist properties

Se estudiaron las propiedades antagonistas del calcio de 2 nuevas 1,4- dihidropiridinas sintéticas (I y II). Ambos compuestos disminuyeron en gran medida las contracciones de anillos de aorta de conejo y con menor potencia inhibieron las contracciones de papilares de ventrículo derecho de rata, acortaron la duración del potencial de acción cardíaco a 0 mV y redujeron la corriente de calcio tipo L en cardiomiocitos ventriculares aislados de rata. La inhibición de la corriente de calcio tipo L fue dependiente del potencial, con un incremento de la potencia de acción en más de un orden de magnitud de concentración en células parcialmente despolarizadas (-50 mV). Estos resultados permiten concluir que las 1,4- dihidropiridinas I y II poseen acciones antagonistas del calcio vasoselectivas marcadas y este mecanismo está dado por el bloqueo de la corriente de calcio tipo L de manera dependiente del potencial(AU)

Biliary excretion of azelnidipine, a calcium antagonist, in rats.

BACKGROUND AND AIMS: Azelnidipine (CS-905) is a novel dihydropyridine calcium antagonist that is known to be excreted in feces. To examine the mechanism of biliary excretion of azelnidipine, the authors studied its biliary excretion in Eisai hyperbilirubinuria rats (EHBR), multidrug resistance protein (Mrp)2-deficient rats, and the effect of cholephilic compounds on the biliary excretion of azelnidipine in rats. METHODS: Radiolabeled azelnidipine was intravenously administered to EHBR and control rats, and the biliary excretion of radiolabeled metabolites was studied. Furthermore, the effect of sulfobromophthalein, taurocholate and vinblastine on the biliary excretion of azelnidipine metabolites was also studied in control rats. RESULTS: The biliary excretion of azelnidipine metabolites was delayed in EHBR. The biliary excretion of azelnidipine metabolites was inhibited by sulfobromophthalein and vinblastine, but was not inhibited by taurocholate or phenothiazine pretreatment. CONCLUSION: These findings suggest that the metabolites of azelnidipine are excreted into the bile partly by Mrp2 and P-glycoprotein.

Lymphocyte calcium signaling involves dihydropyridine-sensitive L-type calcium channels: facts and controversies.

Calcium influx into lymphocytes is essential for activation, differentiation, and effector functions. While several channel- and receptor-types contribute to calcium influx, voltage-gated calcium channels (VGCC) mediate a well-characterized calcium influx pathway that is most exclusively identified in excitable cells. The role of L-type VGCCs, which belong to high-voltage activated calcium channels and are defined as dihydropyridine (DHP) receptors in excitable cells, is well documented. Interestingly, while lymphocytes do not range in the excitable cell category, the modulatory role of DHP agonists and antagonists and the identification of L-type VGCC-related molecules in B and T lymphocytes, mainly in Th2 cells, suggest these proteins are involved in the calcium response of these cells. Because the identity and the regulation of DHP receptors/channels in lymphocytes is far from being solved, we will discuss the challenging issues of demonstrating a role of L-type VGCCs in nonexcitable cells and the arguments supporting their role in lymphocytes. We will comment on the limitation of the use of DHP agonists and antagonists to ascertain a specific involvement of L-type VGCCs in lymphocyte calcium signaling. Finally, we will provide new clues on the interest of a potential use of DHP antagonists in Th2-cell-mediated pathology.

Biophysical properties, pharmacology, and modulation of human, neuronal L-type (alpha(1D), Ca(V)1.3) voltage-dependent calcium currents.

Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming alpha(1) subunit together with several accessory subunits, including alpha(2)delta, beta, and, in some cases, gamma subunits. A family of VDCCs known as the L-type channels are formed specifically from alpha(1S) (skeletal muscle), alpha(1C) (in heart and brain), alpha(1D) (mainly in brain, heart, and endocrine tissue), and alpha(1F) (retina). Neuroendocrine L-type currents have a significant role in the control of neurosecretion and can be inhibited by GTP-binding (G-) proteins. However, the subunit composition of the VDCCs underlying these G-protein-regulated neuroendocrine L-type currents is unknown. To investigate the biophysical and pharmacological properties and role of G-protein modulation of alpha(1D) calcium channels, we have examined calcium channel currents formed by the human neuronal L-type alpha(1D) subunit, co-expressed with alpha(2)delta-1 and beta(3a), stably expressed in a human embryonic kidney (HEK) 293 cell line, using whole cell and perforated patch-clamp techniques. The alpha(1D)-expressing cell line exhibited L-type currents with typical characteristics. The currents were high-voltage activated (peak at +20 mV in 20 mM Ba2+) and showed little inactivation in external Ba2+, while displaying rapid inactivation kinetics in external Ca2+. The L-type currents were inhibited by the 1,4 dihydropyridine (DHP) antagonists nifedipine and nicardipine and were enhanced by the DHP agonist BayK S-(-)8644. However, alpha(1D) L-type currents were not modulated by activation of a number of G-protein pathways. Activation of endogenous somatostatin receptor subtype 2 (sst2) by somatostatin-14 or activation of transiently transfected rat D2 dopamine receptors (rD2(long)) by quinpirole had no effect. Direct activation of G-proteins by the nonhydrolyzable GTP analogue, guanosine 5'-0-(3-thiotriphospate) also had no effect on the alpha(1D) currents. In contrast, in the same system, N-type currents, formed from transiently transfected alpha(1B)/alpha(2)delta-1/beta(3), showed strong G-protein-mediated inhibition. Furthermore, the I-II loop from the alpha(1D) clone, expressed as a glutathione-S-transferase (GST) fusion protein, did not bind Gbetagamma, unlike the alpha(1B) I-II loop fusion protein. These data show that the biophysical and pharmacological properties of recombinant human alpha(1D) L-type currents are similar to alpha(1C) currents, and these currents are also resistant to modulation by G(i/o)-linked G-protein-coupled receptors.

Calcium/calmodulin-dependent protein kinase type IV (CaMKIV) inhibits apoptosis induced by potassium deprivation in cerebellar granule neurons.

The neuroprotective mechanisms of the Ca2+/calmodulin kinase (CaMK) signaling pathway were studied in primary cerebellar neurons in vitro. When switched from depolarizing culture conditions HK (extracellular K+ 30 mM) to LK (K+ 5 mM), these neurons rapidly undergo nuclear fragmentation, a typical feature of apoptosis. We present evidence that blockade of L-type Ca2+ channels (nifedipine sensitive) but not N/P/Q-type Ca2+ channels (omega-conotoxin MVIIC sensitive) triggered apoptosis and CPP32/caspase-3-like activity. The entry into apoptosis was associated with a progressive caspase-3-dependent cleavage of CaMKIV, but not of CaMKII. CaMKIV function in neuronal apoptosis was further investigated by overexpression of CaMKIV mutants by gene transfer. A dominant-active CaMKIV mutant inhibited LK-induced apoptosis whereas a dominant-negative form induced apoptosis in HK, suggesting that CaMKIV exerts neuroprotective effects. The transcription factor CREB is a well-described nuclear target of CaMKIV in neurons. When switched to LK, the level of phosphorylation of CREB, after an initial drop, further declined progressively with kinetics comparable to those of CaMKIV degradation. This decrease was abolished by caspase-3 inhibitor. These data are compatible with a model where Ca2+ influx via L-type Ca2+ channels prevents caspase-dependent cleavage of CaMKIV and promotes neuronal survival by maintaining a constitutive level of CaMKIV/CREB-dependent gene expression.

Reversal of acquired resistance to doxorubicin in K562 human leukemia cells by astemizole.

This study demonstrates that astemizole, a non-sedating anti-histaminergic drug with low toxicity in vivo, greatly potentiates the growth-inhibitory activity of doxorubicin in doxorubicin-resistant human leukemia cells (K562/DXR). Astemizole synergistically potentiated the cytotoxicity of doxorubicin for K562/DXR cells at a concentration of 0.1-3 microM in a dose-dependent manner, whereas they showed hardly any synthergistic effect in the parental cell line (K562) at the same concentration. Since doxorubicin resistance in these cells is associated with the expression of high levels of P-glycoprotein, we evaluated the effect of astemizole on P-glycoprotein activity in cytofluorographic efflux experiments with doxorubicin. Our results indicate that astemizole inhibits the P-glycoprotein pump-efflux activity in a dose-related manner. Moreover, it also inhibits the photolabeling of P-glycoprotein by [3H]azidopine in a dose-dependent manner. These findings provide a biological basis for the potential therapeutic application of astemizole as an anticancer drug either alone or in combination with doxorubicin to multidrug-resistant leukemic cells.

Calcium channels functional roles in the frog semicircular canal.

Different types of voltage-operated calcium channels have been described in hair cells; however, no clear functional role has been assigned to them. As a first functional characterization of vestibular calcium channels, we studied the effect of several calcium channel agonists and antagonists on whole nerve firing rate in an isolated frog semicircular canal preparation. Resting activity was affected by all dihydropyridines tested and by omegaconotoxin GVIA, whereas only nimodipine was able to reduce the mechanically evoked activity. These results indicate that nimodipine-sensitive channels play a major role in afferent transmitter release, and omega-conotoxin GVIA sensitive channels regulate the afferent firing (possibly on the postsynaptic side) but with a less important role.

Identification of the synthetic surfactant nonylphenol ethoxylate: a P-glycoprotein substrate in human urine.

P-glycoprotein (Mdr1p) is an ATP-dependent drug efflux pump that is overexpressed in multidrug-resistant cells and some cancers. Mdr1p is also expressed in normal tissues like the kidney, where it can mediate transepithelial drug transport. A human urinary compound that reverses multidrug resistance and blocks [3H]azidopine photolabeling of P-glycoprotein was purified to homogeneity and identified by 1H-NMR and mass spectrometry as the synthetic surfactant nonylphenol ethoxylate (NPE). Multidrug-resistant Chinese hamster ovary (CHO) C5 cells accumulated less [3H]NPE than parental drug-sensitive Aux-B1 cells, and Mdr1p substrates, verapamil and cyclosporin A, increased this surfactant's accumulation in C5 cells. NPE blocked the net transepithelial transport (basolateral to apical) of [3H]cyclosporin A in epithelia formed by Madin-Darby canine kidney (MDCK) cells. Net transepithelial transport (basal to apical) of [3H]NPE was demonstrated in MDCK cells and was inhibited by cyclosporin A. These findings show NPE is a Mdr1p substrate excreted into urine by kidney P-glycoprotein. NPE is a widely used surfactant and a known hormone disrupter that is readily absorbed orally or topically. The current findings indicate the function of kidney Mdr1p may be to eliminate exogenous compounds from the body.

Neuroprotective activities of carvedilol and a hydroxylated derivative: role of membrane biophysical interactions.

Carvedilol is a vasodilating beta-blocker and antioxidant approved for treatment of mild to moderate hypertension, angina, and congestive heart failure. SB 211475 (4-[2-hydroxyl-3-[[2-(2-methoxyphenoxy)ethyl]amino]propoxyl]-9H-++ +carbazol-3-ol), a hydroxylated carvedilol analogue, is an even more potent antioxidant in several assay systems. Carvedilol also has neuroprotective capacity with modulatory actions at N-methyl-D-aspartate (NMDA) receptors and Na+ channels. In the present study, we demonstrated that in cultured rat cerebellar neurons, SB 211475 has 28-fold greater antioxidant activity than carvedilol, but is 2- to 6-fold less potent, respectively, at inhibiting neurotoxic activities at Na+ channels and at NMDA receptor channels. To determine a biophysical rationale for these differential activities, small angle x-ray scattering data were obtained from model lipid and brain membrane bilayers containing either carvedilol, SB 211475, or dihydropyridine calcium channel blockers. Electron density profiles revealed that the location of SB 211475 was restricted to the glycerol backbone/hydrocarbon interface and significantly reduced membrane width by 5%, whereas the time-averaged location for carvedilol and flunarizine also extended to the hydrated surface of the bilayer. Comparison of carvedilol with several dihydropyridines showed a correlation between high ClogP values (lipophilicity), Na+ channel inhibitory potency, and bilayer localization. The antioxidant activity of SB 211475 could be explained by restricted intercalation into the glycerol phosphate/hydrocarbon interface, creating an increase in volume associated with the phospholipid acyl chains, which would then become resistant to lipid peroxidation. Differential channel modulation may also be explained by these membrane structural results, which indicate that carvedilol and the less spatially restricted dihydropyridine molecules are more likely to inhibit transmembrane receptor channels.

Effect of lacidipine, a dihydropyridine calcium antagonist on renal function of hypertensive patients with renal insufficiency.

There are few studies on the use of dihydropyridine calcium antagonists in hypertensive patients with moderate renal insufficiency. We undertook an open study on the effects on renal function, albumin excretion and blood pressure of the slow-onset, long-acting dihydropyridine calcium antagonist, lacidipine, in 14 patients with stable, chronic renal insufficiency (mean assessed GFR 0.78 ml/s, range 0.50-1.17 ml/s) and moderate hypertension. Following a 2 week washout phase, lacidipine was administered for 24 weeks in a dose of 2 mg/day with the dose being titrated at 2 weekly intervals to a maximum of 6 mg/day in order to achieve adequate blood pressure control. Frusemide was introduced if blood pressure was not controlled on the maximum lacidipine dose. Blood pressure, creatinine clearance, 24 h urinary albumin excretion and plasma creatinine and albumin concentrations were measured at regular intervals throughout the study. Isotopic GFR was determined at the end of the washout period and at week 24. Lacidipine was not very effective in controlling blood pressure and had an adverse effect on renal function. In 3 patients with an incipient nephrotic syndrome this necessitated withdrawal from the study. Mean GFR of the 10 patients who completed the study decreased from 0.69 ml/s/1.73 m2 at baseline to 0.56 ml/s/1.73 m2 at week 24 (p = 0.006) with a decline in GFR being observed in 9 of these patients. The decrease in GFR was greatest in patients with poorly controlled blood pressure. An insignificant increase in mean urinary albumin excretion occurred during the study with this increase being observed only in patients with albuminuria > 1 g/24 h at baseline. These findings indicated that systemic hypertension altered glomerular hemodynamics and that the vasodilatation of pre-glomerular vessels which followed introduction of the calcium antagonist may have exacerbated this situation. The withdrawal of an angiotensin converting enzyme inhibitor during the washout period may have contributed to these changes. We suggest that renal function should be monitored closely in patients with renal insufficiency when a calcium antagonist is being used to control blood pressure, particularly in those with either marginal blood pressure control, significant albuminuria or an incipient nephrotic syndrome.

Lethal drug interactions of sorivudine, a new antiviral drug, with oral 5-fluorouracil prodrugs.

Rats were orally co-administered sorivudine (SRV: 1-beta-D-arabinofuranosyl-(E)-5-(2-bromovinyl)uracil), a new oral antiviral drug for herpes zoster, with the oral anticancer drug tegafur (FT: 1-(2-tetrahydrofuryl)-5-fluorouracil as a prodrug of 5-fluorouracil (5-FU) once daily to investigate a toxicokinetic mechanism of 15 Japanese patients' deaths recently caused within a brief period by the drug interaction of these drugs. All the rats showed extremely elevated levels of 5-FU in plasma and tissues, including bone marrow and small intestine, and died within 10 days, whereas the animals given the same dose of SRV or FT alone were still alive over 20 days without any appreciable toxic symptom. Before their death, there was marked damage of bone marrow, marked atrophy of intestinal membrane mucosa, marked decreases in white blood cells and platelets, diarrhea with bloody flux, and severe anorexia as reported with the Japanese patients. Data obtained by in vivo and in vitro studies strongly suggested that (E)-5-(2-bromovinyl)uracil generated from SRV by gut flora was reduced in the presense of NADPH to a reactive form by hepatic dihydropyrimidine dehydrogenase (DPD), a key enzyme determining the tissue 5-FU levels, bound covalently to DPD as a suicide inhibitor, and markedly retarded the catabolism of 5-FU.

Sympathetic modulation of the effect of nifedipine on myocardial contraction and Ca current in the rat.

The regulation of cardiac L-type Ca2- current (Ica) and contraction by dihydropyridine antagonists and beta-adrenergic receptor agonists has been the subject of numerous studies over the last decade. However, little is known on the crosstalk between these two regulatory pathways. For instance, a fundamental question that remains unanswered is: does activation of the beta-adrenergic receptors modify the sensitivity of the myocardium to dihydropyridine agonists? To answer this question, we examined in the present study how activation of the beta-adrenergic receptors modifies the effects of nifedipine on the mechanical and energetic parameters of the isolated perfused rat heart. Activation of the beta-adrenergic receptors was achieved by perfusing the hearts with isoprenaline, a non-selective beta-adrenergic receptor agonist, and could be reduced by atenolol, a beta-adrenergic receptor antagonist. To examine possible alterations during hypertension in the sensitivity of the hearts to the drug, tested, the study was performed in both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive animals (SHR). While 0.1 microM nifedipine reduced left ventricular pressure (LVP) by 36% and 34% in WKY and SHR rats, respectively, under basal conditions, its effects became negligible in both groups of rats after stimulation of the hearts with 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline restored the inhibitory effect of nifedipine to control values in both WKY and SHR rats. Additional experiments were performed in isolated ventricular myocytes from WKY rats using the whole-cell patch-clamp technique. The inhibitory effects of 0.1 to 1 microM nifedipine were significantly larger on basal Ica than after the current had been previously elevated by 0.1 microM isoprenaline. Addition of 1 microM atenolol in the presence of isoprenaline partially restored the inhibitory effect of nifedipine on Ica. Our results demonstrate a reduced sensitivity of the heart muscle to nifedipine during activation of beta 1-adrenergic receptors. This effect is partly explained by a reduced inhibitory effect of nifedipine on Ic during activation of cAMP-dependent phosphorylation.

Construction of a high-affinity receptor site for dihydropyridine agonists and antagonists by single amino acid substitutions in a non-L-type Ca2+ channel.

The activity of L-type Ca2+ channels is increased by dihydropyridine (DHP) agonists and inhibited by DHP antagonists, which are widely used in the therapy of cardiovascular disease. These drugs bind to the pore-forming alpha1 subunits of L-type Ca2+ channels. To define the minimal requirements for DHP binding and action, we constructed a high-affinity DHP receptor site by substituting a total of nine amino acid residues from DHP-sensitive L-type alpha1 subunits into the S5 and S6 transmembrane segments of domain III and the S6 transmembrane segment of domain IV of the DHP-insensitive P/Q-type alpha1A subunit. The resulting chimeric alpha1A/DHPS subunit bound DHP antagonists with high affinity in radioligand binding assays and was inhibited by DHP antagonists with high affinity in voltage clamp experiments. Substitution of these nine amino acid residues yielded 86% of the binding energy of the L-type alpha1C subunit and 92% of the binding energy of the L-type alpha1S subunit for the high-affinity DHP antagonist PN200-110. The activity of chimeric Ca2+ channels containing alpha1A/DHPS was increased 3.5 +/- 0.7-fold by the DHP agonist (-)Bay K8644. The effect of this agonist was stereoselective as in L-type Ca2+ channels since (+) Bay K8644 inhibited the activity of alpha1A/DHPS. The results show conclusively that DHP agonists and antagonists bind to a single receptor site at which they have opposite effects on Ca2+ channel activity. This site contains essential components from both domains III and IV, consistent with a domain interface model for binding and allosteric modulation of Ca2+ channel activity by DHPs.

Activation of nucleotide receptors inhibits high-threshold calcium currents in NG108-15 neuronal hybrid cells.

UNLABELLED: A P2U (UTP-sensitive) nucleotide receptor has previously been cloned from NG108-15 neuroblastoma x glioma hybrid cells and it has been shown that activation of this receptor inhibits the M-type K+-current. We now report that UTP also inhibits Ca2+-currents in differentiated NG108-15 cells, but probably through a different nucleotide receptor. UTP (100 microM) inhibited the peak of the high-threshold current by 28.4 +/- 3.1% (n = 28) with no effect on the low-threshold current. Two components of high-threshold current were identified: one inhibited by 100 nM omega-conotoxin (CgTx) and one inhibited by 2 microM nifedipine and enhanced by 1 microM BAY K8644. UTP inhibited the former by 31.0 +/- 3.1%, with an IC50 of 2. 8 +/- 1.1 microM, and the latter 34.2 +/- 6.1% with an IC50 of 1.7 +/- 1.3 microM. Pertussis toxin pretreatment prevented inhibition of the CgTx-sensitive, nifedipine-resistant but not CgTx-resistant current. Inhibition was not prevented by intracellular BAPTA (20 mM) or cAMP (1mM). Effects of UTP on both currents were imitated by UDP, ATP, ADP, AP4A and ATPgammaS but weakly or not at all by 2-MeSATP, GTP, AMP-CPP or ITP. Since the receptors which inhibit Ca2+-currents are activated by ATP, it is suggested that they might mediate auto-inhibition of transmitter release by ATP if present on purinergic nerve terminals. KEYWORDS: nucleotides, UTP, ATP, calcium currents, neuroblastoma cells

Catalepsy induced by manidipine, a calcium channel blocker, in mice.

Manidipine, a calcium channel blocker, is a piperazine derivative similar to flunarizine or cinnarizine, which are known to induce parkinsonism. Since it has been reported that manidipine can worsen parkinsonian symptoms in a patient with Parkinson's disease, we have evaluated catalepsy in manidipine-treated mice and compared this with flunarizine-and haloperidol-induced catalepsy. The minimum dose at which manidipine induced catalepsy was 200 times higher than that of haloperidol whereas for flunarizine, the minimum dose was 50 times higher than that for haloperidol. Manidipine, flunarizine and haloperidol occupied both dopamine D1 and D2 receptors and D2-receptor occupancy was higher than D1-receptor occupancy. These results suggest that the blockade of dopamine D1 and D2 receptors by drugs and the drug-induced catalepsy are related to the structure (piperazinyl substituent) of the drugs.

Toxicological comparison of a muscarinic agonist given to rats by gavage or in the diet.

Corneal opacities and urinary tract sepsis were previously observed by the authors in rats given muscarinic agonists mixed in the diet or by gavage. To explain the differential toxicity generated by each means of administration, toxicokinetics of the muscarinic agonist CI-979 were investigated. In addition, the muscarinic antagonist scopolamine was co-administered with CI-979 to evaluate the relationship of these effects to pharmacological mechanism of action of CI-979. Female rats were given CI-979 daily by gavage at 0, 1, 10 and 30 mg/kg body weight or in the diet at 0, 1, 10 and 50 mg/kg body weight for up to 14 days. Dose-related clinical signs of muscarinic stimulation, such as sialorrhoea and dacryorrhoea, were observed predominantly in rats given 10 and 30 mg/kg body weight CI-979 by gavage, and corresponded with the high plasma drug concentrations. In contrast, hydronephrosis, pyelonephritis, and inflammation and necrosis of the kidney, urinary bladder, urethra and urinary papilla were linked to sustained, albeit lower plasma drug concentrations attained by dietary administration of CI-979 at 10 and 50 mg/kg body weight. Comparable incidences of corneal opacities were induced by both means of administration, but lesions appeared more rapidly and were generally of greater severity when CI-979 was given in the diet. The induction of corneal lesions, as well as urinary sepsis, may not relate simply to maximum plasma concentrations or to areas under the curve per se, but rather may arise when plasma drug concentrations are sustained. Corneal opacification and development of urinary tract pathology were inhibited by scopolamine, suggesting that these effects were related to the muscarinic mechanism of action of CI-979.

Pharmacological analysis of voltage-dependent potassium currents in cultured skeletal myocytes of the frog Rana temporaria.

Previously, the existence of nine types of outward potassium current (IK) was shown. The whole family of IK may be divided into two groups: fast transient currents (f) with time to peak less than 70 ms (at test potential near 0 mV), and slow (s) components (Lukyanenko et al. 1993). The latter were completely blocked by 4-aminopyridine (4-AP) and the former were more sensitive to TEA than slow IK. In the present study we analyzed the effects of calcium blockers on different types of IK using the whole-cell patch-clamp technique. One to seven-day-old myocytes without slow calcium current and without contact with nerve cells were examined. Extracellullar application of 40-80 mumol/l dihydropyridine (DHP) antagonist nifedipine did not change maximal conductance of K-channels, but induced a parallel shift by 5-10 mV of chord conductance curve along the voltage axis in the direction of more negative potentials. Quinidine in concentrations 30-200 mumol/l caused a reversible block of the fast and the slow IK (C0.5 = 75 mumol/l), and enhanced the current decay (2-3-fold at 150 mumol/l). Verapamil (VP) in concentrations 100-700 mumol/l reduced IK with dose-dependent effect (C0.5 = 200 mumol/l) and changed its kinetic properties. VP 100 mumol/l caused a complete irreversible block of the slow IK. VP reduced the time inactivation constant of fast IK with a dose-dependent effect (8-10-fold at 300 mumol/l), and this effect was stronger during depolarizing pulses. The latter points to the possibility that the fast K-channels preferentially bind VP in open state. An analysis of the effects suggests that K-channels of the frog myocytes could be divided into 2 groups: 1) K-channels which irreversibly blocked by VP and 4-AP (slow), and 2) those reversibly inhibited by VP and 4-AP (fast potassium channels).