Effects of dihydropyridine calcium channel antagonists in ethanol withdrawal; doses required,stereospecificity and actions of Bay K 8644

The effects of dihydropyridine calcium channel antagonists, and the calcium channel activator, Bay K 8644, were examined on the convulsive behaviour induced by handling in mice following withdrawal from chronic ethanol inhalation. Nimodipine and nitrendipine and PN 200-110 significantly decreased the convulsive behaviour, after intraperitoneal doses of the same order of magnitude as have been found by others to be required for displacement of radiolabelled dihydropyridine in the CNS. The (+) isomer of PN 200-110 was effective, but the (-) isomer, which is ineffective in vitro, had no significant action. Bay K 8644 prevented the actions of nimodipine against the ethanol withdrawal syndrome. The behavioural ratings after nimodipine plus Bay K 8644 were significantly higher than after vehicle treatment. Bay K 8644 alone, when given to naive mice, caused convulsive behaviour resembling that seen in withdrawal from chronic ethanol treatment, but when given during ethanol withdrawal did not significantly increase the behavioural signs.     

Effects of the calcium channel activator Bay K 8644 on general anaesthetic potency in mice.

1. The effects of the calcium channel activator, Bay K 8644, on the anaesthetic potencies of ethanol, argon and pentobarbitone were examined in mice. 2. Bay K 8644, at 1 mg kg-1 i.p., significantly antagonized the general anaesthetic potencies of ethanol and argon, but at 5 and 10 mg kg-1 this compound increased the general anaesthetic potency of these drugs. 3. At doses of 1, 5 and 10 mg kg-1 Bay K 8644 antagonized the anaesthetic effects of pentobarbitone. Bay K 8644, at the highest dose used, did not alter the brain concentrations of pentobarbitone or the blood concentrations of ethanol. 5. The effects of the different doses of Bay K 8644 on the actions of ethanol and of argon are compatible with the known partial agonist properties of this compound on calcium channels in vitro. 6. The actions of Bay K 8644 on the anaesthetic effects of pentobarbitone suggests that specific interactions may be involved in the anaesthetic actions of this compound.     

Interactions between diltiazem and ethanol: differences from those seen with dihydropyridine calcium channel antagonists

It has previously been shown that dihydropyridine calcium channel antagonists prevent the ethanol withdrawal syndrome and potentiate the acute effects of ethanol and other central depressant drugs. We now report that, in contrast, the benzothiazepine calcium channel antagonist, diltiazem, gave no protection against the behavioural hyperexcitability seen during ethanol withdrawal, when given either acutely, on withdrawal, or chronically, during the ethanol treatment. A significant increase in convulsive behaviour on handling was seen during the withdrawal period when diltiazem was given on cessation of a mild chronic ethanol treatment schedule. Diltiazem decreased the acute general anaesthetic effects of ethanol, and did not appear to potentiate the ataxic action of ethanol. Centrally administered diltiazem did not enhance the hypothermic action of ethanol, but this effect was significantly increased by diltiazem when the calcium channel antagonist was given peripherally. When given alone by the intraperitoneal route, diltiazem decreased spontaneous locomotor activity and lowered body temperature. When the intracerebral route was used for administration of diltiazem, a significantly decrease in body temperature was seen when this compound was given alone, accompanied by a brief hyperexcitability. The interactions between ethanol and diltiazem therefore appear to differ from those seen with other calcium channel antagonists.     

Chronic dihydropyridine treatment can reverse the behavioural consequences of and prevent adaptations to, chronic ethanol treatment.

1. Chronic treatment with the dihydropyridine calcium channel antagonist, nitrendipine, given concurrently with ethanol, prevented the ethanol withdrawal syndrome in mice, even though the chronic nitrendipine treatment was stopped 24 h or 48 h before the withdrawal testing. 2. This effect was seen in two strains of mice with different methods of ethanol administration. Nitrendipine was effective when given for two weeks but not after only two days' treatment. 3. Two other dihydropyridine calcium antagonists, nimodipine and PN 200-110, given chronically with ethanol, also prevented the withdrawal syndrome. The tests were again made 24 h after the last administration of dihydropyridine. 4. The chronic nitrendipine treatment also prevented the rise in the number of central dihydropyridine binding sites that occurs on chronic ethanol administration. 5. Chronic administration of nitrendipine alone did not cause any withdrawal behaviour. 6. Chronic nitrendipine treatment did not affect the seizure threshold to bicuculline in mice that were not given ethanol. 7. Whole brain concentration measurements showed that the effects were not due to residual nitrendipine in the CNS at the time of withdrawal testing or to differences in central ethanol concentrations during the treatment. 8. It is suggested that the results provide evidence for a functional role for dihydropyridine-sensitive calcium channels in ethanol dependence.     

Dihydropyridine receptors in the pregnant human uterus in vitro

The stimulatory effect of the dihydropyridine derivative, Bay K 8644, on the isolated pregnant human uterus, and its interactions with the calcium channel blockers, nifedipine, verapamil and diltiazem and with the calmodulin inhibitor trifluoperazine were investigated. In uterine preparations showing spontaneous activity, Bay K 8644 (1 nmol/l-1 mumol/l) produced an increase in the frequency of contractions without effects on their amplitude. However, strong phasic contractions were induced in quiescent preparations. The stimulatory action of Bay K 8644 proved to be insensitive to calcium withdrawal, but was completely prevented in the presence of 1 mmol/l EGTA. Bay K 8644 shifted the inhibitory concentration-response curve of verapamil and nifedipine to the right, leaving the diltiazem- and trifluoperazine-induced effect virtually unchanged. Schild plot analysis revealed a competitive interaction of Bay K 8644 with nifedipine, while the interaction with verapamil was of the nonlinear type. These data demonstrated that the dihydropyridine derivative Bay K 8644 possesses calcium agonistic properties also in the isolated human uterus. Furthermore, the competitive interaction with nifedipine showed the existence of specific dihydropyridine receptors closely associated with the calcium channel.     

Voltage-dependent effects of YC-170, a dihydropyridine calcium channel modulator,in cardiovascular tissues

Voltage-dependent effects of YC-170, a putative calcium channel activator, were examined and compared with those of Bay K 8644 in isolated guinea-pig cardiac tissues and rabbit aortae. In guinea-pig left atria superfused with a normal bathing solution (4 mmol/l K+), both YC-170 (10 mumol/l) and Bay K 8644 (1 mumol/l) produced a positive inotropic action accompanied by a prolongation of action potential durations (APDs). In normally-polarized guinea-pig papillary muscles Bay K 8644 increased force of contraction (fc) and APDs. However, YC-170 failed to increase fc in spite of a slight prolongation of APDs. In papillary muscles partially depolarized by 25 mmol/l K+ solution, Bay K 8644 enhanced the electrically-induced slow action potentials and contractile force. In contrast with Bay K 8644, YC-170 significantly depressed the slow action potentials and decreased fc. YC-170 also showed the depressant action on the slow action potentials induced by isoproterenol (0.1 mumol/l), histamine (3 mumol/l) and tetraethylammonium (10 mmol/l) plus high Ca2+ (4 mmol/l). In sinoatrial node cells of guinea-pig right atria Bay K 8644 produced a positive chronotropic action with increases in the maximum rate of rise (Vmax) and action potential amplitude (APA), whereas YC-170 produced a negative chronotropic action with decreases in Vmax and APA. In the rabbit aortic strips preincubated with bathing solution containing various concentrations of K+ (15, 20, 30 and 40 mmol/l), Bay K 8644 produced concentration-dependent contractions in a range of concentrations up to 0.3 mumol/l. However, when the concentration exceeded 1 mumol/l, Bay K 8644 caused a slight relaxation, irrespective of the K+ concentrations of bathing solution. YC-170 in concentrations of 10 and 30 mumol/l contracted the aortic strips placed in 5.9 or 15 mmol/l K+ bathing solution, but caused relaxation in 30 or 40 mmol/l K+ bathing solution. These results suggest that YC-170 is a dihydropyridine calcium channel modulator which behaves as a Ca2+ channel agonist in tissues of high membrane potentials, but as a Ca2+ channel antagonist in tissues of low membrane potentials.     

Nitrendipine, given during drinking, decreases the electrophysiological changes in the isolated hippocampal slice, seen during ethanol withdrawal.

1. Extracellular recordings were made from mouse isolated hippocampal slices prepared after chronic treatment in vivo with either ethanol or ethanol plus the dihydropyridine calcium channel antagonist, nitrendipine. 2. The withdrawal of ethanol caused a variety of changes in the field potentials, as previously reported, including decreases in the thresholds for eliciting single and multiple population spikes, increases in paired pulse potentiation and shifts to the left of the input/output curves. 3. The addition of nitrendipine to the drinking mixture in the chronic ethanol treatment significantly decreased all the changes in the field potentials that were seen after ethanol withdrawal. 4. Addition of nitrendipine to the perfusion medium also decreased the signs of hyperexcitability seen in the hippocampal slices during ethanol withdrawal. 5. The results provide further evidence that neuronal calcium channels may be involved in ethanol dependence and that the adaptive changes caused by chronic ethanol treatment can be modulated by alterations at dihydropyridine-sensitive sites.     

Effects of intracoronary Bay k 8644, a calcium channel agonist, in anesthetized dogs

Bay k 8644, a new dihydropyridine calcium channel activator has been shown to have positive inotropic and vasoconstrictor properties following intravenous (i.v.) administration. In the present study, intracoronary administration of Bay k 8644 was used to isolate drug effects on regional myocardial blood flow and contractility independent of systemic hemodynamic actions in beta-adrenoceptor-blocked anesthetized dogs. Intracoronary infusion of Bay k 8644 (1.5, 3.7, 7.4, 14.8 micrograms/min) produced significant increases in contractility (percentage of segment shortening) in the drug-perfused region. Peak positive dP/dt, an index of global contractility, was increased in parallel with increases of regional contractility. No other changes in systemic hemodynamics occurred. Transmural tissue blood flow distribution as measured by radioactive microspheres was also unchanged by Bay k 8644. Intracoronary infusion of KB-944, a nondihydropyridine calcium channel blocking agent, increased coronary blood flow and decreased regional segment shortening and peak positive dP/dt. KB-944 inhibited increases in contractility produced by Bay k 8644. Thus, Bay k 8644 was shown to have a direct positive inotropic effect in vivo which was inhibited by calcium channel blockade.     

Differential interactions between benzodiazepines and the dihydropyridines, nitrendipine and Bay K 8644.

The effects of the dihydropyridine calcium antagonist, nitrendipine and the calcium channel activator, Bay K 8644, have been compared on the anaesthetic, ataxic and anticonvulsant effects of benzodiazepines. Possible interactions between the peripheral benzodiazepine receptor antagonist, PK11195, and the classical benzodiazepines were also examined. Nitrendipine considerably potentiated the anaesthetic effects of benzodiazepines and increased their ataxic effects but had no effect on the anticonvulsant actions. Clonazepam did not produce anaesthesia, at doses up to 1 g kg-1 or when given with nitrendipine. When given alone, nitrendipine did not cause general anaesthesia. Nitrendipine did not appear to alter the metabolism of midazolam. The calcium channel activator, Bay K 8644, reduced the anaesthetic potency of midazolam and, when given alone, produced ataxia. It did not significantly alter central concentrations of midazolam. The "peripheral" benzodiazepine antagonist, PK11195, did not affect the ataxic or anaesthetic actions of benzodiazepines. These results suggest that dihydropyridine-sensitive calcium channels may be more important to the general anaesthetic than to the anticonvulsant actions of benzodiazepines. The "peripheral" benzodiazepine site did not appear to play a role in either of these properties.     

Different antioxidative potencies of dihydropyridine calcium channel modulators in various models

There is evidence that dihydropyridine calcium antagonists (DHP) play a beneficial role during the development of atherosclerosis. Since antioxidative properties of this substance class may be important, we investigated the antioxidative potency of the DHP prototype calcium channel antagonist nifedipine, the long acting calcium channel antagonist lacidipine, the DHP calcium channel agonist Bay K 8644 and the bulky DHP derivate Bay O 5572 (negligible effects on L-type calcium channels) in three different models. Additionally, we examined the potential correlation between lipophilic and antioxidative properties. In an in vitro model, Bay K 8644 was significantly more effective in scavenging superoxide anions (hypoxanthine/xanthine-oxidase-assay) than lacidipine, Bay O 5572 or nifedipine (micro- to millimolar concentration range). Addition of artificial membrane preparations (dimyristoylphosphatidylcholine) to mimic a physiological environment resulted in an enhanced antioxidative effect, with lacidipine being the most effective DHP to quench radicals (low micromolar concentration range). Thirdly, in a more physiological model of hyperglycemia (30 mmol/l) induced release of reactive oxygen species (ROS) from native endothelial cells of porcine coronary arteries, we showed that nifedipine was a significantly more potent antioxidant (therapeutical nanomolar concentration range) than the other DHP. Calculation of the lipophilicity of the four substances (lacidipine>Bay O 5572>Bay K 8644>nifedipine) showed a positive correlation between the antioxidative potency and the lipophilicity in the model with the artificial membranes but not in the other models. We conclude that it seems necessary to access antioxidative properties of substances in physiological models in which we could demonstrate that nifedipine exhibits ROS-quenching properties in a therapeutic concentration range.     

Ethanol withdrawal seizures and the NMDA receptor complex

Prior biochemical and electrophysiological studies have shown that low doses of ethanol inhibited calcium influx through the N-methyl-D-aspartate (NMDA) receptor/ionophore. The present data show that chronic ethanol treatment results in an increase in the number of NMDA receptor/ionophore complexes in the hippocampus, a brain area known to be associated with ethanol withdrawal seizure activity. Treatment during withdrawal with NMDA-exacerbated handling induced withdrawal seizures in the ethanol-dependent mice, while administration of the NMDA receptor-associated calcium channel antagonist MK-801 decreased the occurrence and severity of the withdrawal seizures in a dose-dependent manner. The results are consistent with the hypothesis that the up-regulation of the NMDA receptor systems following chronic ethanol treatment may mediate the seizures associated with ethanol withdrawal in dependent animals.     

Bay K 8644 potentiates the anxiolytic effect of ethanol.

The possible role of voltage-sensitive calcium channels (VSCCs) in the anxiolytic effect of ethanol was investigated using three different doses of ethanol (0.5, 1.0 and 2.0 g/kg) with calcium agonist Bay K 8644 (0.5 mg/kg) and calcium antagonist nifedipine (5 mg/kg) in rats. Ethanol produced an anxiolytic effect in a dose-dependent manner. The Bay K 8644-potentiated anxiolytic effect of ethanol, however, Bay K 8644 did not alter anxiety when used alone. Nifedipine itself showed an anxiolytic effect but did not change the ethanol-induced anxiolytic effect. This finding may lead to the consideration of the neurochemical mechanisms of the anxiolytic effects of ethanol and nifedipine as they vary from each other.     

Effects of diltiazem in convulsive states differ from those previously reported for dihydropyridine calcium channel antagonists

Unlike the dihydropyridine calcium channel antagonists studied previously, the benzothiazepine calcium channel antagonist, diltiazem, increased the incidence of convulsions caused by bicuculline,N-methyl-DL-aspartate or 4-aminopyridine. However, the latencies to convulsions were also increased. Diltiazem increased the ratings of convulsive behaviour on handling after intraperitoneal administration of bicuculline, or pentyl-enetetrazol and after the calcium channel activator, Bay K 8644, administered ICV. When the binding of the dihydropyridine, [³H]-nitrendipine in the CNS was measured in vivo, this was increased by diltiazem. This compound therefore showed a different pattern of interaction with convulsant drugs then that previously demonstrated for other calcium channel antagonists, appearing to possess both pro- and anticonvulsant actions, and a different pattern of interaction with the dihydropyridine receptor complex.     

Calcium channel agonist and antagonist binding in a highly enriched sarcolemma preparation obtained from canine ventricle

The purpose of the present study was to characterize the binding of the Ca2+ channel agonist (+/-)[3H]Bay K 8644 in a highly enriched cardiac membrane preparation and to examine its interactions with other Ca2+ channel ligands. Scatchard analysis showed that (+/-)[3H]Bay K 8644 displayed some 265-fold less affinity for its receptor than did the Ca2+ channel antagonist (+)[3H]PN200-110. Furthermore, (+/-)[3H]Bay K 8644 binding site density was significantly less than that seen for (+)[3H]PN200-110. Kinetic analysis of (+/-)[3H]Bay K 8644 binding revealed biphasic association and dissociation rates. The Ca2+ channel antagonist MDL 12,330A stimulated both (+)[3H]PN200-110 and (+/-)[3H]Bay K 8644 binding by effects on Kd. In contrast, diltiazem inhibited (+)[3H]PN200-110 binding but had no effect on (+/-)[3H]Bay K 8644 binding. Both MDL 12,330A and diltiazem inhibited Ca2+-dependent contraction in rat aortic rings but the combination of these two drugs was less than additive in this regard. We conclude that the binding profile of (+/-)[3H]Bay K 8644 results from the racemic nature of this ligand and that MDL 12,330A and diltiazem interact at sites distinct from one another on or around the slow voltage-dependent Ca2+ channel.     

A calcium channel antagonist stereoselectively decreases ethanol withdrawal hyperexcitability but not that due to bicuculline, in hippocampal slices.

1. Extracellular recordings were made from CA1 area of isolated hippocampal slices of the mouse after chronic ethanol administration in vivo, with orthodromic stimulation of the Schaffer collateral/commissural fibres. 2. The (+)-isomer of the calcium channel antagonist PN 200-110 (isradipine) significantly decreased all the recorded signs of hyperexcitability in the slices during ethanol withdrawal. These included increased paired pulse potentiation and decreases in the thresholds for elicitation of single and multiple population spikes. 3. The (-)-isomer of PN 200-100 did not affect ethanol withdrawal hyperexcitability in the slices. 4. Neither isomer of PN 200-110 affected the field potentials in slices from control animals. 5. The gamma-aminobutyric acid (GABA) antagonist, bicuculline, lowered thresholds for eliciting population spikes in hippocampal slices from untreated animals. The active, (+)-isomer of PN 200-110 did not affect this action of bicuculline in hippocampal slices from untreated animals. 6. The stereoisomerism of the action of PN 200-110 on ethanol withdrawal hyperexcitability in the hippocampal slice was therefore the same as that seen in blockade of calcium channels. The results suggested that ethanol withdrawal hyperexcitability recorded in the isolated hippocampal slice involved increased activity of voltage-sensitive calcium channels.     

The differential effects of felodipine and nitrendipine on cerebral dihydropyridine binding ex vivo and the ethanol withdrawal syndrome in mice.

1. The ability of two dihydropyridine calcium channel antagonists, felodipine and nitrendipine both to displace [3H]-isradipine binding in CNS tissue measured ex vivo and to protect against the ethanol withdrawal syndrome has been investigated. 2. Mice were injected with various doses of felodipine or nitrendipine and [3H]-isradipine binding measured in brain homogenates prepared 0.5, 3 or 5 h later. Inhibition versus dose curves were sigmoid and the dose required to produce 50% inhibition increased linearly with time after administration. Felodipine was approximately 10 times more potent than nitrendipine. 3. Nitrendipine (50 mg kg-1, i.p.) and felodipine (10 mg kg-1, i.p.) produced around a 75% inhibition of [3H]-isradipine binding 3 h later. Binding of [3H]-nitrendipine to cerebral tissues measured after in vivo injection of the ligand was decreased by nitrendipine (50 mg kg-1) and felodipine (10 mg kg-1) to a similar extent. 4. Nitrendipine (50 mg kg-1) prevented the behavioural signs of ethanol withdrawal as measured by handling induced convulsions, but felodipine (10 mg kg-1 or 2 mg kg-1) did not provide any protection against this effect of ethanol withdrawal. Felodipine (10 mg kg-1, twice daily) during the course of ethanol treatment also failed to attenuate the withdrawal syndrome. 5. The convulsive response to a mild audiogenic stimulus during ethanol withdrawal was increased following one dose of felodipine (5 mg kg-1, i.p.) but unaffected by nitrendipine. 6. Injection of Bay K 8644 (60 microgram, i.c.v.) produced a significant increase in handling-induced convulsive behaviour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration-related differences in the effects of the Ca2+ agonist Bay K 8644 in human bronchus in vitro

1. In human isolated bronchial muscle Bay K 8644 (1 nM-1 microM) increased the force of contraction. In concentrations greater than 1 microM (in sensitized tissues), and 10 microM (in control), Bay K 8644 relaxed tissues to below their pretreatment baseline tension. 2. In control tissues contracted with methacholine or LTD4, Bay K 8644 (10 microM) produced a further small contraction; Bay K 8644 (100 microM) relaxed the tissues below baseline. 3. In tissues contracted with histamine, Bay K 8644 (10-100 microM) produced only relaxation. 4. Bay K 8644 therefore has both Ca2+ agonist and Ca2+ antagonist properties, the calcium agonist activity is more pronounced in sensitized tissues.     

Effects of verapamil and Bay K 8644 on defibrillation energy requirements in dogs.

Antiarrhythmic drugs are often required in patients with implantable cardioverter-defibrillator devices. Prior evidence suggests that drugs modulate defibrillation energy requirements by altering ion channel activity. To evaluate the effects of calcium ion channel activity on internal defibrillation energy requirements, the calcium antagonist verapamil and Bay K 8644, a calcium channel activator, were investigated in 30 open-chest, pentobarbital-anesthetized dogs. Defibrillation energies were applied across two epicardial patch electrodes. The likelihood of successful defibrillation was determined at various shock energy levels, and the 50 and 90% effective energy doses were calculated using nonlinear regression. In saline control experiments (n = 10), the stability of the preparation throughout the 6-h duration of the experiments could be demonstrated. Verapamil administration (n = 10) infused to a mean plasma concentration of 69 ng/ml increased the 50 and 90% effective defibrillation energies by 41 and 43% (p less than 0.05), respectively, and to a mean plasma verapamil concentration of 170 ng/ml by 95 and 75% (p less than 0.01), respectively. The mean cycle length during ventricular fibrillation decreased with verapamil and was inversely related to the change in defibrillation energy requirement. Administration of Bay K 8644 (n = 10) produced a slight increase in the 50% effective defibrillation energy (25%; p less than 0.05) and 90% effective defibrillation energy (17%; n.s.). The electrophysiologic effects of verapamil were neither prevented nor reversed by Bay K 8644. In conclusion, intravenous verapamil administration caused an increase in defibrillation energy requirements, but the mechanism by which verapamil exerted this effect remains unclear. These experimental data suggest that verapamil should be used in patients with automatic implantable cardioverter-defibrillator devices only after individual testing.     

Dihydropyridine Bay K 8644 activates T lymphocyte calcium-permeable channels

The effects of the dihydropyridine calcium channel agonist Bay K 8644 on indo-1-loaded Jurkat human leukemia T lymphocytes was assessed by flow cytometry. Bay K 8644 from 10(-9) to 10(-4) M caused a dose-dependent rise in the intracellular free Ca concentration, an effect that was not mimicked by the dihydropyridine Ca antagonist nifedipine. Single channel recordings by the extracellular patch-clamp technique indicated that Bay K 8644 activated an 8-pS, barium-permeable channel that opened as bursts of brief events. The channel appeared to be identical to the previously described voltage-insensitive, messenger-mediated, calcium-permeable channel involved in T cell activation. The predominant effect of Bay K 8644 on these channels was to increase the probability of channel reopening, apparently without a major effect on mean channel open-time. The results suggest that the dihydropyridine Ca agonist Bay K 8644 interacts with both voltage-gated and receptor-operated Ca channels and also suggest potential strategies for development of a new class of immunomodulatory drugs.     

Calcium antagonist properties of diclofurime isomers. I. Functional aspects

Trans-diclofurime has been shown to be a potent calcium antagonist which resembles verapamil in vitro and in vivo. Trans-diclofurime was a potent antagonist of Ca2+-induced contractions in K+-depolarized taenia preparations from the guinea pig caecum (pA2 = 8.3 +/- 0.2), whereas the cis isomer was 50 times less active (pA2 = 6.6 +/- 0.1); the inhibitory effects of trans-diclofurime were reversed noncompetitively by the Ca2+ channel activator Bay K 8644. Trans-diclofurime and verapamil were equipotent inhibitors of electrically evoked contractions of guinea pig left atria preparations; the inhibitory effects were frequency dependent and cis-diclofurime was 10 times less effective. Both diclofurime isomers prolonged the effective refractory period at high concentrations, indicating that they also possess local anaesthetic properties. Trans-diclofurime and verapamil reduced blood pressure in pithed rats infused with angiotensin II. Hypotensive effects were accompanied by bradycardia and prolongation of PR intervals, leading to second-degree atrioventricular block. The cis isomer was less potent. Diclofurime is thus a very potent calcium antagonist in heart and smooth muscle and has some additional membrane-stabilizing properties.