Finasteride, a 5alpha-reductase inhibitor, potentiates antinociceptive effects of morphine, prevents the development of morphine tolerance and attenuates abstinence behavior in the rat

It has been shown that morphine increases 5alpha-reductase enzyme activity in the rat central nervous system; however importance of this finding on morphine analgesia, tolerance and dependence has not been reported. In the present study, we investigated inhibition of 5alpha-reductase enzyme on morphine effects using finasteride. To determine whether the 5alpha-reductase enzyme interact with morphine analgesia, finasteride (5 mg/kg, i.p.) was administrated with morphine (5 and 7 mg/kg, i.p.). The tail-flick test was used to assess the nociceptive threshold, before and 15, 30, 45, 60 and 90 min after drug administration. In tolerance experiments, morphine 20 mg/kg was injected i.p., twice daily for 4 days. The development and expression of dependence were assessed in the naloxone precipitation test 5 days after the morphine (20-30 mg/kg, i.p.) administration. We found that finasteride could potentiate the antinociceptive effect of morphine. In addition, chronic finasteride administration effectively blocked development of tolerance and dependence to morphine. Following chronic morphine administration, single dose injection of finasteride failed to reverse tolerance but prevented naloxone precipitate withdrawal syndrome. Therefore, it was concluded that there is a functional relationship between 5alpha-reductase enzyme and morphine.     

Chronic social defeat stress-induced enhancement of T-type calcium channels increases burst-firing neurons in the ventral subiculum

The ventral subiculum (vSub), a representative output structure of the hippocampus, serves as a main limbic region in mediating the brain's response to stress. There are three subtypes of subicular pyramidal neurons based on their firing patterns: regular-spiking (RS), weak-bursting (WB) and strong-bursting (SB) neurons, located differently along proximal–distal axis. Here, we found that chronic social defeat stress (CSDS) in mice increased the population of SB neurons but decreased RS neurons in the proximal vSub. Specific blockers of T-type calcium channels inhibited the burst firings with a concomitant reduction of afterdepolarization, suggesting that T-type calcium channels underlie the burst-spiking activity. Consistently, CSDS increased both T-type calcium currents and expression of Cav3.1 proteins, a subtype of T-type calcium channels, in the proximal vSub. Therefore, we conclude that CSDS-induced enhancement of Cav3.1 expression increased bursting neuronal population in the vSub, which may contribute to stress-related behaviors.     

T型钙通道在神经病理性痛模型中介导疼痛的研究进展

神经病理痛是临床上常见病症,其发病机制尚不清楚,目前尚无有效的治疗手段,其慢性神经病理痛持续时间长,故其研究成为疼痛领域的热点和重点。近年来发现T型钙通道在神经病理性疼痛中起到了关键性的作用。本文将近年T型钙通道在神经病理性痛模型中介导疼痛的机制研究进展加以综述。     

Synthesis of some new C2 substituted dihydropyrimidines and their electrophysiological evaluation as L-/T-type calcium channel blockers

Drugs targeting different calcium channel subtypes have strong therapeutic potential for future drug development for cardiovascular disorders, neuropsychiatric diseases and cancer. This study aims to design and synthesize a new series of C2 substituted dihydropyrimidines to mimic the structure features of third generation long acting dihydropyridine calcium channel blockers and dihydropyrimidines analogues. The target compounds have been evaluated as blockers for Ca_V1.2 and Ca_V3.2 utilizing the whole-cell patch clamp technique. Among the tested compounds, compound 7a showed moderate calcium channel blockade activity against Ca_V3.2. Moreover, the predicted physicochemical properties and pharmacokinetic profiles of the target compounds recommend that they can be considered as drug-like candidates. The results highlight some significant information for the future design of lead compounds as calcium channel blockers.     

N型钙通道与疼痛

N型电压依赖性钙通道(VDCCs)在疼痛的传递与调控中具有重要作用。它们密集分布于脊髓背角伤害感受性神经元突触前末梢,参与主要疼痛介质如谷氨酸和P物质等释放的调节。通过阻断上述通道,选择性N型VDCCs阻断剂表现出强效镇痛作用,N型VDCCs Cav2．2亚基基因敲除小鼠也表现为痛阈提高。N型VDCCs还分布于自主神经系统和中枢神经系统突触部位,现有的N型VDCCs阻断剂用于疼痛治疗时出现的各种副作用与这些部位的突触抑制有关。最近发现,背根节伤害感受性神经元上存在一种特异的N型VDCCs亚型,这为疼痛治疗提供了一个非常有意义的新靶标。     

No evidence of morphine analgesia to noxious shock in the shore crab, Carcinus maenas

A number of criteria have been suggested for testing if pain occurs in animals, and these include an analgesic effect of opiates (Bateson, 1991). Morphine reduces responses to noxious stimuli in crustaceans but also reduces responsiveness in a non-pain context. Here we use a paradigm in which shore crabs receive a shock in a preferred dark shelter but not if they remain in an unpreferred light area. Analgesia should thus enhance movement to the preferred dark area because they should not experience ‘pain’. However, morphine inhibits rather than enhances this movement even when no shock is given. Morphine produces a general effect of non-responsiveness rather than a specific analgesic effect and this could also explain previous studies claiming analgesia. However, we question the utility of this criterion of pain and suggest instead that behavioural criteria be employed.     

Effect of age, vitamin D, and calcium on the regulation of rat intestinal epithelial calcium channels

Transepithelial transport of calcium involves uptake at the apical membrane, movement across the cell, and extrusion at the basolateral membrane. Active vitamin D metabolites regulate the latter two processes by induction of calbindin D and the plasma membrane ATPase (calcium pump), respectively. The expression of calbindin D and the calcium pump declines with age in parallel with transepithelial calcium transport. The apical uptake of calcium is thought to be mediated by the recently cloned calcium channels-CaT1 (or ECaC2, TRPV6) and CaT2 (or ECaC1, TRPV5). The purpose of these studies was to determine whether there were age-related changes in intestinal calcium channel regulation and to identify the dietary factors responsible for their regulation. Young (2 months) and adult (12 months) rats were fed either a high calcium or low calcium diet for 4 weeks. The low calcium diet significantly increased duodenal CaT1 and CaT2 mRNA levels in both age groups, but the levels in the adult were less than half that of the young. The changes in calcium channel expression with age and diet were significantly correlated with duodenal calcium transport and with calbindin D levels. To elucidate the relative roles of serum 1,25(OH)2D3 and calcium in the regulation of calcium channel expression, young rats were fed diets containing varying amounts of calcium and vitamin D. Dietary vitamin D or exogenous 1,25(OH)2D3 more than doubled CaT1 mRNA levels, and this regulation was independent of dietary or serum calcium. These findings suggest that the apical calcium channels, along with calbindin and the calcium pump, may play a role in intestinal calcium transport and its modulation by age, dietary calcium, and 1,25(OH)2D3.     

The role of calcium channels in substance P-induced contractile response in the rat iris

This study was undertaken to assess the role of calcium channels in the contractile response induced by substance P in the isolated rat iris. Substance P produced graded and sustained contraction in the rat iris. Pre-incubation of preparations with thapsigargin (1 μM), verapamil (1 μM), isradipine (1 μM) or with ω-conotoxin MCIIA (0.1 μM) did not significantly inhibit substance P-mediated contraction in the isolated rat iris. However, pre-incubation of the preparations with nicardipine (1 μM) or ruthenium red (1 mM) caused parallel displacement to the right of the substance P concentration–response curve without affecting its maximal response. In contrast, amiloride (1 μM), markedly inhibited substance P-mediated contraction (73±5%), while econazole (1 mM) also significantly inhibited (44±11%) substance P-mediated contraction in the isolated rat iris. Collectively, these results suggest that substance P-mediated contractile response in the isolated rat iris depends largely on the influx of external Ca²⁺, by a mechanism which might involve the T-type calcium channels.     

Proteasome inhibitor lactacystin disturbs the intracellular calcium homeostasis of dopamine neurons in ventral mesencephalic cultures

Ubiquitin proteasome system (UPS) impairment has been implicated in the pathology of Parkinson's disease, but the mechanisms underlying the UPS impairment-induced dopamine (DA) neuron degeneration remain obscure. To test whether calcium homeostasis disturbance is involved in the DA neuronal injury resulting from UPS impairment, we treated the primary ventral mesencephalic (VM) cultures with the proteasome inhibitor lactacystin, and observed its effects on the expression of the gene Homer 1a that is related to calcium homeostasis, and the intracellular free calcium ([Ca²⁺]_i) levels as well as the DA neuron survival. We also investigated a possible role of the L-type voltage dependent calcium channels (L-VDCC) in these events. We found that the lactacystin exposure induced the Homer 1a expression, lowered the [Ca²⁺]_i levels, reduced the depolarization-induced calcium entry and DA release in the VM cultures, and caused a significant DA neuron loss. Activation of L-VDCC by potassium chloride or its agonists alleviated the effects of lactacystin on the [Ca²⁺]_i levels and promoted DA neuron survival, whereas L-VDCC antagonists blocked the depolarization-mediated neuroprotective effect, and at high concentrations the L-VDCC antagonists aggravated the lactacystin-induced DA neuronal injury. These results indicate that calcium homeostasis disturbance may be a novel pathological mechanism leading to DA neuronal injury under conditions of proteasome inhibition.     

Stimulation of voltage-dependent calcium channels during capacitation and by progesterone in human sperm

To fertilize, mammalian sperm must undergo two sequential steps that require activation of calcium entry mechanisms, capacitation and acrosomal exocytosis, induced in the latter case by the egg zona pellucida glycoprotein ZP3 or by progesterone. Voltage-dependent calcium channels (VDCC) could participate in these processes. Since patch clamp recordings are extremely difficult in mature sperm, the activity of VDCC has been alternatively analyzed with optical detectors of membrane potential and intracellular calcium in sperm populations. Using this approach, we previously reported that in human sperm there is a voltage-dependent calcium influx system that strongly indicates that human sperm are endowed with functional VDCC. In this study we developed evidence indicating that calcium influx through VDCC is significantly stimulated during sperm in vitro capacitation and by progesterone action, which is present in the follicular fluid that surrounds the egg. The observed effects of capacitation and progesterone on VDCC may be physiologically significant for sperm-egg interaction.     

Impact of phosphomimetic and non‐phosphorylatable mutations of phospholemman on L‐type calcium channels gating in HEK 293T cells

Background : Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L‐type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods : The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild‐type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. Results : WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage‐dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM‐induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Conclusions : Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects.     

The effect of Cyclin‐dependent kinase 5 on voltage‐dependent calcium channels in PC12 cells varies according to channel type and cell differentiation state

Cyclin‐dependent kinase 5 (Cdk5) is a Ser/Thr kinase that plays an important role in the release of neurotransmitter from pre‐synaptic terminals triggered by Ca²⁺ influx into the pre‐synaptic cytoplasm through voltage‐dependent Ca²⁺ channels (VDCCs). It is reported that Cdk5 regulates L‐, P/Q‐, or N‐type VDCC, but there is conflicting data as to the effect of Cdk5 on VDCC activity. To clarify the mechanisms involved, we examined the role of Cdk5 in regulating the Ca²⁺‐channel property of VDCCs, using PC12 cells expressing endogenous, functional L‐, P/Q‐, and N‐type VDCCs. The Ca²⁺ influx, induced by membrane depolarization with high K⁺, was monitored with a fluorescent Ca²⁺ indicator protein in both undifferentiated and nerve growth factor (NGF)‐differentiated PC12 cells. Overall, Ca²⁺ influx was increased by expression of Cdk5‐p35 in undifferentiated PC12 cells but suppressed in differentiated PC12 cells. Moreover, we found that different VDCCs are distinctly regulated by Cdk5‐p35 depending on the differentiation states of PC12 cells. These results indicate that Cdk5‐p35 regulates L‐, P/Q‐, or N‐type VDCCs in a cellular context‐dependent manner.

     

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.     

The role of calcium ions in gravity singal perception and transduction

Ca²⁺ is implicated as a messenger in coupling various environmental stimuli, such as gravity and light, to response. In recent years, it has become evident that Ca²⁺ plays a central role in all three phases of gravitropism – perception, transduction and response. The root cap, which is known to contain high amounts of Ca²⁺ and calmoduin, is the primary site of gravity preeception. The possible role of phosphoinositide turnovr and Ca²⁺ and Ca²⁺ calmodulin-dependent enzymes such as Ca²⁺– ATPase and protein kinases in gravitropsim is discussed. A model is proposed to describe the role of Ca²⁺ in both normal and light-dependnt gravity response in roots.     

Differential development of acute tolerance to analgesia, respiratory depression, gastrointestinal transit and hormone release in a morphine infusion model

To determine whether the differences in development of acute tolerance to several morphine actions correlate with the mu receptor subtype mediating them, we have examined the appearance of acute tolerance to analgesia, respiratory depression, gastrointestinal transit, and hormone release in an intravenous morphine infusion model. Analgesia, a naloxonazine-sensitive mu1 action, peaked at 2 hr after initiation of the infusions. The log dose-response relationship of the infusion rate to peak tailflick latency was linear from 10 to 50 micrograms/kg/min. By 8 hr, the tailflick latencies declined nearly to baseline levels, implying the rapid development of tolerance. Tolerance to morphine-induced prolactin release, another mu1 action, also developed rapidly over 8 hr. In contrast two mu2 actions, respiratory depression measured with arterial blood gas, determinations and gastrointestinal transit, showed no significant tolerance over a similar 8 hr infusion. We also observed no tolerance to morphine-induced growth hormone release, a non-mu1 action, over the same period. Thus, these results demonstrate that mu1 actions develop tolerance in an infusion model far more rapidly than a number of naloxonazine-insensitive (non-mu1) ones and may help explain differences in the rate of tolerance development to morphine actions.     

The biochemical activation of T-type Ca2+ channels in HEK293 cells stably expressing alpha1G and Kir2.1 subunits

In order to investigate the currently unknown cellular signaling pathways of T-type Ca(2+) channels, we decided to construct a new cell line which would stably express alpha(1G) and Kir2.1 subunits in HEK293 cells (HEK293/alpha(1G)/Kir2.1). Compared to cells which only expressed alpha(1G) (HEK293/alpha(1G)), HEK293/alpha(1G)/Kir2.1 cells produced an enormous inward rectifying current which was blocked by external Ba(2+) and Cs(+) in a concentration-dependent manner. The expression of Kir2.1 channels contributed significantly to the shift of membrane potential from -12.2+/-2.8 to -57.3+/-3.7mV. However, biophysical and pharmacological properties of alpha(1G)-mediated Ca(2+) channels remained unaffected by the expression of Kir2.1 subunits, except for the enlarging of the window current region. Biochemical activation of alpha(1G) channels using 150mM KCl brought about an increase in [Ca(2+)](i), which was blocked by mibefradil, the T-type Ca(2+) channel blocker. These data suggest that the HEK293/alpha(1G)/Kir2.1 cell line would have potential uses in the study of T-type Ca(2)(+) channel-mediated signaling pathways and possibly useful in the development of new therapeutic drugs associated with T-type Ca(2)(+) channels.     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.     

The role of calcium in stalk development and in phosphate acquisition in Caulobacter crescentus

Calcium was found to stimulate stalk development in Caulobacter crescentus and to relieve the inhibition of development long known to be caused by phosphate. This suggested that phosphate inhibition could be attributed to its interaction with Ca²⁺, thereby depriving the cells of a factor that promoted development. Calcium was also found to promote phosphate acquisition by the cells, observed as acceleration of growth at extremes of phosphate concentration, as promotion of carbon-source utilization rather than storage, and as support for phosphate-dependent resistance to arsenate inhibition of growth. Cytological studies of dividing cells revealed that stalked siblings had greater access to exogenous phosphate for use in growth or for storage as polyphosphate, and that access of non-stalked sibling to phosphate was dependent on the length of the stalk of the dividing cell. It was concluded that the physiologic role of the stalk is enhancement of phosphate acquisition. The stimulatory role of calcium in this process was attributed to its support of stalk development and to its stabilization of internal membrane/cell envelope association within the cell-stalk juncture.Abbreviations EGTA (ethyleneglycol-bis-(-aminoethyl ether)-N,N-tetraacetic acid) - PHB (poly--hydroxybutyric acid) - Pn (inorganic polyphosphate) This report is dedicated to the memory of an outstanding teacher, Roger Y. Stanier. If he were available to evaluate this work, I could be confident of his providing the most incisive criticism; if not convinced, the reason(s) for his dissatisfaction would be made quite clear, and if convinced, his defense undoubtedly would enlarge my understanding of this microorganism