钙通道阻滞剂抗肝缺血—再灌注损伤作用机制的实验研究

目的 探讨钙通道阻滞剂（ＣＣＥＢ）对肝缺血一再灌注损伤（ＨＩＲＩ）防治作用的机制。方法制备家兔ＨＩＲＩ模型,动态观察维拉帕米（ＶＰ）和地尔硫卓（ＤＴ）对肝组织及血中黄嘌呤氧化酶（ＸＯ）、超氧化物歧化酶（ＳＯＤ）活性及脂质过氧化物（ＬＰＯ）浓度的影响。结果 ＶＰ组和ＤＴ组ＯＸ活性及ＭＤＡ含量分别显著低于对照组（均Ｐ＜０．０１）,而ＳＯＤ活性与对照组比较均无显著性差异（均 Ｐ＞ ０．０５）。结论 ＣＣＥＢ抗 ＨＩＲＩ的机制与其降低 ＸＯ活性、抑制脂质过氧化反应密切相关。     

Intracellular mechanisms of verapamil and diltiazem action on striated muscle of the rabbit

Summary Skinned fibres from striated muscle were used to study the intracellular mechanisms (contractile proteins and sarcoplasmic reticulum [SR]) of action of diltiazem (DT) and verapamil (VP) on muscle contraction. Rabbit papillary muscle (PM), and the skeletal muscles adductor magnus (AM, fast-twitch) and soleus (SL, slow-twitch) were used. The muscles were skinned by homogenization and fibre bundles for PM and single fibres for AM and SL were dissected from the homogenate and mounted on photodiode force transducers.VP (0.1–3.0 mmol/l) (and to a lesser degree DT) increased Ca²⁺ -activated tension development of the contractile protains in PM and SL and decreased it in AM (+[4–20]%, +4%, –[14–28]%, respectively). Both drugs increased the submaximal Ca²⁺ -activated tension development at the order of PM = SL > AM in a dose-dependent manner. The changes of half-maximal pCa₅₀ at 1 mmol/l VP were 0.25, 0.25, and 0.15, respectively.For Ca²⁺ uptake and release from the SR, VP as well as DT (0.1–3.0 mmol/l) in the uptake phase decreased caffeine-induced tension transients in a dose-dependent fashion. At 0.01–3.0 mmol/l, the drugs directly induced Ca²⁺ release from the SR or enhanced caffeine-induced tension transients with the exception that in PM, DT attenuated caffeine-induced tension transients.Thus, VP and DT have similar intracellular mechanisms of action in striated muscle. Both drugs induce calcium release from the SR and increase Ca²⁺ sensitivity of the contractile proteins, and thus could be the underlying mechanisms for potentiating twitch tension, and inducing contracture in skeletal muscle. Send offprint requests to J. Y. Su at the above address     

Antioxidant response and biocompatibility of curcumin-loaded triblock copolymeric micelles

To evaluate the safety profile of cationic micelles, based on triblock copolymer poly(dimethylaminoethyl methacrylate)–poly(e-caprolactone)–poly(dimethylaminoethyl methacrylate) (PDMAEMA₉– PCL₇₀–PDMAEMA₉), the effects of empty (PM) and curcumin loaded micelles (PM-Curc) on nonenzyme induced lipid peroxidation (LPO) in vitro, hemolytic activity and morphological changes in some organs after repeated intraperitoneal administration in vivo were studied. To induce LPO, rat liver microsomes were incubated with a solution of iron sulfate and ascorbinic acid (Fe²⁺/AA). The effect of empty PM (40 and 100?μg/ml), PM-Curc and free curcumin (both at 3.48 and 8.7?μg curcumin/ml) was assessed at 20?min incubation time. In the non-enzyme induced LPO model, the investigated substances at all concentrations significantly decreased the formation of malondialdehyde (MDA), compared to the Fe²⁺/AA induced LPO group. According to the results it can be concluded that curcumin alone and loaded in PM, exert significant antioxidant activity. In the biocompatibility safety studies, the mean hemolytic index for polymeric carrier was less than 2%, indicating it was non-hemolytic. The general appearance of the organ tissues from Wistar rats, treated in vivo with curcumin loaded PM was similar to that of controls, thus showing no apparent toxicity after repeated 14-days treatment.     

Effects of mood-stabilizing drugs on the peripheral serotonin transporter

We studied the effect of lithium (L), carbamazepine (CBZ), valproic acid (VA), verapamil (VP) and nifedipine (NF) on the specific binding of ³H-imipramine (³H-IMI) to platelet membranes, as compared with clomipramine (CLO). The results showed that VP, NF and CLO exerted a concentration-dependent inhibition on the IMI binding; CLO had the most potent inhibitory effect. No effect was observed with L. CBZ or VA. These results suggest that while VP and NF interact with the 5-HT transporter complex, L, CBZ and VA do not. Therefore, their effective (or suggested) mood-stabilizing effects are likely to be related to different mechanisms.     

Effects of Calcium Blockers on the Cytosolic Calcium,H2O2 Production and Elastase Release in Human Neutrophils

Abstract: Activated neutrophils are assumed to be one plausible cause of tissue injury in the ischaemic and reperfused myocardium. We studied the inhibitory effects of the calcium antagonists felodipine, nimodipine and verapamil on human neutrophil activation in order to elucidate the mechanisms underlying their myocardioprotective effects and to determine whether calcium antagonists with different chemical structures vary in their effect on neutrophil activation. Neutrophils were stimulated with formyl-Met-Leu-Phe (0.1 μM) or by phorbol myristate acetate (0.16 μM), and the rise in cytosolic calcium and the H₂O₂ production were determined. For felodipine, the inhibitory effect on granulocyte elastase release was also studied. The calcium antagonists reduced formyl-Met-Leu-Phe and phorbol myristate acetate-induced neutrophil activation in a concentration-dependent manner, the order of potency being: felodipine >nimodipine >verapamil. For felodipine, the IC₅₀ (concentration causing 50% reduction) values were 3×10^?6 and 2×10^?6 M for the formyl-Met-Leu-Phe-induced cytosolic calcium increase and H₂O₂ production, respectively. The IC₅₀ -value for the phorbol myristate acetate-induced cytosolic calcium increase was 6×10^?6 and for H₂O₂ production 4×10^?6 M. For formyl-Met-Leu-Phe-induced granulocyte elastase release, the IC₅₀-value was 5×10^?6 M. The inhibitory effect of felodipine on the phorbol myristate acetate-induced granulocyte elastase release did not exceed 50%. Nimodipine was a less potent inhibitor than felodipine for both formyl-Met-Leu-Phe- and phorbol myristate acetate-induced cell activities. Verapamil was even less potent than the other two agents. The present study demonstrates that felodipine potentially suppresses neutrophil activation at micromolar concentrations. However, this observation should not be directly extrapolated to explain the tissue protection by the compounds without evidence of profound local accumulation.     

Increased Ca2+ sensitivity of myofibrillar tension in ischaemic vs dilated cardiomyopathy

1. There is evidence that different aetiologies of heart failure, especially ischaemic vs dilated cardiomyopathy (ICM and DCM, respectively), may influence the prognosis of patients with this disease. Patients with ICM have a worse prognosis than those with DCM; the mechanisms underlying this difference have not yet been clarified. The aim of the present study was to investigate whether there are changes in myofibrillar function depending on the aetiology of human heart failure. 2. Ca²⁺‐dependent tension (DT) and actomyosin ATPase acitivity (MYO) in Triton X‐skinned fibre preparations of the left ventricular myocardium from patients with heart failure due to ICM (n = 5) and DCM (n = 5) were measured. Tension‐dependent ATP consumption was calculated by the ratio of DT and MYO (‘tension cost’). Non‐failing myocardium (NF) from donor hearts, which could not be transplanted because of technical reasons, was evaluated as a control. 3. Although DT was reduced, the myofibrillar Ca²⁺ sensitivity of DT and MYO, as well as tension cost, were increased in preparations from ICM and DCM myocardium compared with NF. The Ca²⁺ sensitivity of DT and MYO was significantly increased in ICM compared with DCM preparations, resulting in more economic cross‐bridge cycling in ICM than in DCM. 4. In conclusion, ICM is associated with an increased Ca²⁺ sensitivity of myofibrillar tension and ATPase activity accompanied by decreased tension cost compared with DCM. Thus, the worse prognosis associated with ICM does not seem to be due to differences in myofibrillar function.     

Facilitation of the vasorelaxant action of calcium antagonists by basal nitric oxide in depolarized artery

We investigated the effect of NO/cyclic GMP pathway on the action of calcium antagonists (isradipine, nisoldipine, lacidipine, verapamil, diltiazem) in rat aorta exposed to 100 mM KCl. For this purpose constitutive NO synthase was blocked by using 100 M N-nitro-l-arginine (l-NNA).The steady-state contractile response evoked by 100 mM KCl was enhanced when the basal NO release had been blocked. The combined effects of basal NO release and calcium antagonists resulted in an inhibition greater than additive. Concentrations of calcium antagonists producing 50% inhibition of contraction were about 3-fold lower in the presence of the basal NO release than in its absence (P < 0.01). ⁴⁵Ca^2– influx stimulated by 100 mM KCl was not affected by the basal NO release, but was inhibited by isradipine and verapamil regardless of NO blockade. Thus, the facilitation of the action of calcium antagonists by NO/cyclic GMP pathway seemed not to be accompanied by a modification of their action on L-type calcium channels. To confirm this, we measured the contractile tension and the calcium signal in fura-2 loaded rings, pretreated with either verapamil or verapamil plus 8-bromo cyclic GMP (BrcGMP), and further exposed to increasing concentrations of extracellular Ca^2– ([Ca²⁺]_o) in 100 mM KCl solution. The increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) evoked by increasing ([Ca²⁺]_o) in rings pretreated with verapamil alone was not different from rings pretreated with verapamil plus BrcGMP. In contrast, the [Ca²⁺]_o-contraction curve was significantly shifted to the right in rings pretreated with verapamil plus BrcGMP.These results show that the NO/cyclic GMP pathway facilitates the inhibitory effect of calcium antagonists on 100 mM KCl-evoked contraction. This phenomenon is not related to a modification of calcium channel blockade, but could result from the reduction of the sensitivity of contractile machinery to Ca²⁺ by cyclic GMP.     

Calcium channel involvement in magnetic field inhibition of morphine-induced analgesia

Summary An exposure for 60 min to a weak 0.5 Hz rotating magnetic field significantly reduced the day-time analgesic effects of morphine in male mice. The dihydropyridine (DHP) calcium channel antagonists diltiazem and nifedipine and the non-DHP antagonist verapamil, as well as the inorganic calcium channel blockers, La³⁺ and Co²⁺, differentially reduced, while the DHP calcium channel agonist, BAY K 8644, enhanced the inhibitory effects of the magnetic stimuli. In a similar manner, though to a lesser degree, the calcium channel antagonists and agonist, increased and decreased, respectively, the inhibitory effects of intracerebroventricular administrations of Ca²⁺ on morphine-induced analgesia. The calcium channel antagonists and agonists had no significant effects on naloxone-mediated reductions of morphine-induced analgesia. These results suggest that exposure to magnetic stimuli affects the functioning of calcium channels and the distribution of calcium ions, thereby, altering the effects of opiates. Send offprint requests to M. Kavaliers     

Inhibition of rat cardiac calcium pump activity by cationic amphiphilic drugs

Cationic amphiphilic drugs such as imipramine, chlorimipramine, chlorpromazine, chlorphentermine, and chloroquine have been shown to cause myocardial toxicity including arrhythmia, cardiomyopathy, and myocarditis. Since calcium transport by sarcoplasmic reticulum (SR) plays a critical role in contraction-relaxation coupling of myocardium, the toxicity exhibited by these drugs could be due to their effects on this phenomenon. Hence, we have studied the effects in vitro and in vivo of imipramine, chlorimipramine, chlorpromazine, chloroquine, and chlorphentermine on cardiac SR ⁴⁵Ca-uptake and Ca²⁺-ATPase in male Fisher-344 rats. For in vitro studies, cardiac SR was prepared from normal rat hearts. For in vivo studies, rats were treated with drugs by oral gavage (p.o) at a dose of 20 pair-fed. All these drugs in vitro inhibited the cardiac SR ⁴⁵Ca-uptake to a varying extent. The order of potency is chlorimipramine > chlorpromazine > imipramine > chlorphentermine > chloroquine. The inhibitory pattern of these drugs on cardiac SR Ca²⁺-ATPase also followed the same trend. The chlorinated analogs of these drugs are more potent in inhibiting ⁴⁵Ca-uptake as well as Ca²⁺-ATPase as compared to their nonchlorinated con-geners. In vivo, all the drugs except chloroquine inhibited the ⁴⁵Ca-uptake and Ca²⁺-ATPase activity to a different extent. The inhibition of cardiac SR calcium pump activity was seen as early as 7 days of treatment with chlorimipramine whereas all the other drugs (imipramine, chlorpromazine, and chlorphentermine) inhibited this calcium pump only at 21 days of treatment. Although in vivo chloroquine-induced inhibition of calcium pump activity did not reach significant level, it seems that there may be an effect at higher concentrations. In summary, the present results indicate that these cationic amphiphilic drugs might be exerting myocardial toxicity due to changes in cellular Ca²⁺ homeostasis because of their effects on cardiac SR calcium transport phenomenon. © 1992 Wiley-Liss, Inc.     

Modification of skeletal muscle sarcotubular Ca2+-stimulated adenosine triphosphatase activity by various agents.

Ca²⁺ -stimulated ATPase was isolated from the sarcotubular fraction of rabbit skeletal muscle, and the effects of various interventions on its activity were studied in the presence of different concentrations of calcium. Decreasing the pH of the incubation medium from 7.5 to 6.0 or the concentration of K⁺ from 100 to 40 mM decreased the enzyme activity. Different anions (I^?, SCN^?, Br^? and NO₃^?) and cations (Hg²⁺, La³⁺ Ni²⁺, Sr²⁺, Mn²⁺ and Co²⁺) were found to inhibit Ca²⁺-stimulated ATPase activity. Ca²⁺ antagonists (verapamil and D600) and β-adrenergic blocking agents (propranolol and practolol) also had inhibitory effects whereas cyclic AMP-protein kinase augmented the enzyme stimulation by calcium. These results implicate sarcotubular Ca²⁺ -ATPase as a possible site of action of various agents affecting the skeletal muscle function.     

In-vitro Negative Chronotropic and Inotropic Effects of a Novel Dihydropyridine Derivative,CD-832, in the Guinea-pig: Comparison with Calcium-channel Antagonists

The effects of CD-832 (4R-(-)-2-(nicotinoylamino)ethyl-3-nitroxypropyl-1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5-pyridine dicarboxylate), a novel dihydropyridine derivative, on guinea-pig isolated myocardial preparations have been compared with those of Ca²⁺-channel antagonists. All ten compounds induced concentration-dependent negative chronotropic effects on preparations of isolated right atria and negative inotropic effects on isolated right ventricular papillary muscles. The order of potency for the negative chronotropic effect was CD-832 > nicardipine = gallopamil > clentiazem > nifedipine = efonidipine > amlodipine = semotiadil > verapamil > diltiazem; that for the negative inotropic effect was nicardipine = gallopamil > nifedipine > verapamil > CD-832 > diltiazem > clentiazem > efonidipine = semotiadil > amlodipine. The ratio of the EC50 (the concentration of Ca²⁺ antagonist having 50% of the maximum effect) for the negative inotropic effect divided by the EC50 for the negative chronotropic effect, considered to be an index of selectivity for negative chronotropic effect, was higher for CD-832, amlodipine, efonidipine and semotiadil than for the other Ca²⁺ antagonists. The ratio for CD-832, nifedipine, nicardipine, efonidipine, amlodipine, verapamil, gallopamil, diltiazem, clentiazem and semotiadil was 11·4, 0·29, 0·87, 35·4, 37·1, 0·65, 0·87, 0·92, 7·11 and 30·0, respectively. These findings indicate that CD-832 and the newly developed Ca²⁺ antagonists including amlodipine, efonidipine, semotiadil and clentiazem were selective for a negative chronotropic effect rather than for a negative inotropic effect. This ‘chrono-selective’ effect of these drugs might be of benefit in the treatment of cardiovascular disorders.     

Different Mechanism of Relaxation Induced by Aporphine Alkaloids in Rat Uterus

Abstract— We have examined the uterine relaxant action of three aporphine molecules (S-glaucine, S-boldine and R-apomorphine) in two experimental conditions, with and without calcium in the bathing solution, and compared these effects with those obtained with the calcium antagonists verapamil and diltiazem. The present study shows that the alkaloids relax the uterine muscle but with different mechanisms of action. In Ca²⁺-containing solution all three alkaloids relaxed the uterus previously contracted by KCl or acetylcholine, but in Ca²⁺-free medium only R-apomorphine was able to relax oxytocin-induced contraction. The calcium antagonists, verapamil and diltiazem, relaxed KCl- or acetylcholine-induced contraction in Ca²⁺-containing solution, whereas they only relaxed oxytocin-induced contraction in Ca²⁺-free medium at much higher doses. These results suggest that glaucine and boldine behave as specific calcium entry blockers without affecting the contractile machinery or intracellular Ca²⁺ levels as apomorphine does. The absolute configuration (S-glaucine and S-boldine vs R-apomorphine) may account for this different action.     

Role of oxidative stress, mitochondrial membrane potential, and calcium homeostasis in human lymphocyte death induced by nickel carbonate hydroxide in vitro

When isolated human lymphocytes were treated in vitro with various concentrations of soluble form of nickel carbonate hydroxide (NiCH) (0–1 mM), at 37°C for 4 h, both concentration- and time-dependent effects of NiCH on lymphocyte death were observed. Increased generation of hydrogen peroxide (H₂O₂), superoxide anion (O ₂ ⁻ ), depletion of both no protein (NP-) and protein (P-) sulfhydryl (SH) contents and lipid peroxidation (LPO) were induced by NiCH. Pretreatment of lymphocytes with either catalase (H₂O₂ scavenger), or deferoxamine (DFO) (iron chelator), or excess glutathione (GSH) (an antioxidant) not only significantly reduced the NiCH-induced generation of H₂O₂ and LPO, but also increased the NP-SH and P-SH contents initially reduced by NiCH. NiCH-induced generation of excess O ₂ ⁻ but not excess LPO was significantly reduced by pretreatment with superoxide dismutase (SOD). NiCH-induced lymphocyte death was significantly prevented by pre-treatment with either catalase, or dimethylthiourea/mannitol (hydroxyl radical scavengers), or DFO, or excess GSH/N-acetylcysteine. NiCH-induced lymphocyte death was also significantly prevented by pretreatment with excess SOD. Thus, various types of oxidative stresses play an important role in NiCH-induced lymphocyte death. Cotreatment with cyclosporin A (a specific inhibitor of alteration in mitochondrial membrane potential (ΔΨ_m) not only inhibited NiCH-induced alteration in ΔΨ_m, but also significantly prevented Ni-compound-induced lymphocyte death. Furthermore, NiCH-induced destabilization of cellular calcium homeostasis. As such, NiCH-induced lymphocyte death was significantly prevented by modulating intracellular calcium fluxes such as Ca²⁺ channel blockers and intracellular Ca²⁺ antagonist. Thus, the mechanism of NiCH (soluble form)-induced activation of lymphocyte death signalling pathways involves not only the excess generation of different types of oxidative stress, but also the induction of alteration in ΔΨ_m and destabilization of cellular calcium homeostasis as well.     

Inhibition of calcium uptake and catecholamine release by 8-(n,n-diethylamino)- octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) In cultured bovine adrenal chromaffin cells

Effects of intracellular calcium antagonists, 8-(f,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8) and 1-(5-(p-nitrophenyl)-furfurylidene-amino) hydantoin sodium hydrate (dantrolene sodium), on catecholamine release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. TMB-8 inhibited carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner with a similar potency. On the contrary, dantrolene sodium did not show obvious inhibitory effects of catecholamine release and ⁴⁵Ca²⁺ uptake. Although TMB-8 inhibited the high K⁺-evoked catecholamine release and ⁴⁵Ca²⁺ uptake, the potency of the drug was approximately 100-fold less than when used to inhibit the carbamylcholine-evoked catecholamine release and ⁴⁵Ca²⁺ uptake. The inhibitory effect of TMB-8 on the carbamylcholine-evoked catecholamine release was not overcome by an increase in an extracellular calcium concentration, and was not due to competitive antagonism at the nicotinic receptor site. Moreover, TMB-8 inhibited the carbamylcholine-stimulated ⁴⁵Ca²⁺ efflux, but dantrolene sodium failed to affect it. These results suggest that TMB-8, a well-known intracellular calcium antagonist, prevents the cellular calcium uptake in cultured adrenal chromaffin cells, and thus prevents catecholamine release.     

The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range

The role of ATP-stimulated P2X₁ receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y₁ or P2Y₁₂ receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X₁ receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X₁ antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on meATP (P2X₁ receptors)-induced or ADP (P2Y₁ receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y₁ & P2Y₁₂ receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8,8,8-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X₁ antagonist reported to date, blocking meATP-induced Ca²⁺ increases and shape change in a concentration-dependent manner, with a pA₂ of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X₁ receptor displayed the following order : NF864>NF449NF110>NF023=MK-HU1=suramin. A different antagonistic profile was observed for ADP-induced Ca²⁺ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X₁ responses, with the order NF110>NF449NF864MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X₁ receptors in thrombosis and haemostasis, NF864 being the most promising compound.     

Compared effects of calcium entry blockers on calcium-induced tension in rat isolated cerebral and peripheral resistance vessels

Summary The effects of the calcium entry blockers verapamil (V), diltiazem (D), nifedipine (NF) and nicardipine (NC) have been studied on calcium concentration-effect curves elicited in depolarized (K⁺, 40 mmol/l) and in serotonin-exposed (6 mol/l) rat middle cerebral arteries (RMCA) in order to compare the relative potencies of the blockers against these two calcium channel activating mechanisms. In control conditions, Ca²⁺ sensitivity expressed as pD₂ and maximal active wall tension (AWT) were not significantly different in depolarized and in 5-HT-exposed vessels: pD₂: 3.39 ±0.08 vs 3.50 ± 0.06 and AWT: 0.93 ± 0.15 mN · mm^–1 vs 0.90 ± 0.16 mN · mm^–1 respectively. V, D, NF and NC displaced Ca²⁺ control curves to the right and depressed the maximum contractile response in the two experimental conditions, which suggests a noncompetitive type of antagonism. All the blockers were more potent inhibitors of Ca²⁺-induced contractions in depolarized than in serotonin-exposed middle cerebral arteries. The IC₅₀ values (concentration of blockers producing a 50% inhibition of maximal control contractile response) were (nmol/l) : V = 20, D = 120, NF = 0.4, NC = 1 and V = 400, D = 10000, NF = 20, NC = 7 in depolarized and serotonin-exposed arteries respectively. From these IC₅₀ values, the relative order of potency of the CEB's was not the same in the two experimental conditions suggesting that while serotonin and K⁺ both promote the entry of Ca²⁺ into vascular smooth muscle cells of RMCA, they either activate a different gating mechanism associated with a single common channel or perhaps distinct channels. Comparison of the results obtained in this study for depolarized rat middle cerebral arteries with those previously obtained in depolarized rat mesenteric resistance arteries (RMRA) revealed that while Ca²⁺-induced contractile responses were inhibited in a similar non-competitive manner by the four CEB's, the respective IC₅₀ values showed that potencies and rank of relative potency of the blockers were different in the two types of vessels. D and NC were equally potent in both preparations (IC₅₀ ratio = 2.5 and 3 respectively) but RMCA were more sensitive to V and NF than RMRA (IC₅₀ ratio = 6.5 and 11 respectively). These results are discussed and it is proposed that regional differencies in the conformation and/or the activation of the voltage-gated Ca²⁺ channels may exist in different vascular beds. Send offprint requests to J. L. Freston     

Antidepressant activity and calcium signaling cascades

Although antidepressant treatments produce clear effects on monoaminergic neuronal function, the link between these effects and therapeutic response to treatment is controversial. Previous studies have demonstrated that antagonists of the NMDA receptor‐gated calcium ionophore result in antidepressant‐like responses in rodents and humans. Likewise, antidepressant treatments produce regionally selective adaptation of the NMDA receptor suggestive of diminished capacity to gate calcium into receptive neurons. Similarly, voltage‐dependent calcium channel antagonists have been reported to produce antidepressant‐like effects in rodents. A major target of increases in subcellular calcium concentration is nitric oxide synthase (NOS) which liberates NO in response to stimulation. Recently, we have demonstrated that nitric oxide synthase antagonists produced antidepressant‐like response in both in vivo preclinical screening procedures and in post‐mortem in vitro studies of β‐adrenoceptor density. We propose: 1) that interruption of the Ca²⁺‐calmodulin‐NOS‐guanylyl cyclase subcellular signaling pathway at any point will produce antidepressant‐like effects; 2) that the acute actions of antidepressants in preclinical screening procedures are a consequence of their ability to disrupt Ca²⁺‐calmodulin‐NOS‐guanylyl cyclase signaling; 3) that chronic but, not acute treatment with antidepressants results in adaptation of the Ca²⁺‐calmodulin‐NOS‐guanylyl cyclase signaling pathway; 4) that this adaptation is necessary for the achievement of the therapeutic actions of antidepressants and; 5) that major depression is accompanied by an alteration (hyperactivity?) of subcellular Ca²⁺ signaling. Copyright © 2001 John Wiley & Sons, Ltd.     

Characterization of the muscarinic receptor mediating contraction of the dog prostate

1 We have studied the effects of muscarinic cholinoceptor agonists and subtype-preferring antagonists on the isometric contraction of smooth muscle strips from dog prostate. 2 Acetylcholine and carbachol induced contraction of prostate strips from the peripheral zone, (‘the capsule’). Bethanechol contracted the tissue but not at lower doses. McN-A-343 and oxotremorine-M showed the same effects. 3 Blocking α- and β-adrenoceptors with phentolamine and propranolol, respectively, did not modify carbachol-induced contractions. 4 The nicotinic receptor blocker, hexamethonium (10^??6–10^??4 m ) did not affect the contractile response evoked by a single dose of carbachol (10^??5 m ), whilst the muscarinic receptor antagonist, atropine (10^??11–10^??9 m ), inhibited it in a competitive manner. 5 The muscarinic M₁ (pirenzepine), M₂[AF-DX 116, himbacine (M₂/M₄) and methoctramine], M₃ (HHSID and f-F-HHSID), and putative M₄ (tropicamide) antagonists reduced significantly the carbachol-induced contractions. The pIC₅₀ values were: atropine (10.01) > himbacine (8.3) > methoctramine (7.85) > AF-DX 116 (7.60) > HHSID (7.21) > p-F-HHSID (7.10) > pirenzepine (7.30) > tropicamide (7.00). 6 The antagonist profile indicates that an predominant M₂ receptor subtype could mediate the muscarinic contraction in the canine prostate.     

Pharmacological profile of NOP receptors coupled with calcium signaling via the chimeric protein Gαqi5

In this study, the Gα_qi5 protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca²⁺ pathway in CHO cells. [Ca²⁺]_i levels were monitored using the fluorometer FlexStation II and the Ca²⁺ dye Fluo 4 AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured: naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Gα_qi5–NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II–Gα_qi5–NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists.     

Formation of alginate microspheres produced using emulsification technique

This study investigated the formative process of alginate microspheres produced using an emulsification technique. The alginate microspheres were produced by cross-linking alginate globules dispersed in a continuous organic phase using various calcium salts: calcium chloride, calcium acetate, calcium lactate and calcium gluconate. The size, shape, drug content and Ca²⁺ content of the microspheres were evaluated. The tack, viscosity and pH of the calcium salt solution and percentage of Ca²⁺ partitioned into the organic phase were determined. Microscopic examination of the test emulsion at various stages of the emulsification process was also carried out. The propensity of cross-linking reaction was found to be dependent on successful collision between alginate and calcium salt globules. Examination of the characteristics of microspheres indicated that the formed microsphere was a resultant product of alginate globule clustering. The growth propensity of microspheres was promoted by the higher rate and extent of cross-linkage which was governed by the pH, tack and/or Ca²⁺ content of the cross-linking solution, as well as the dissociation constant and diffusivity of the calcium salt. Overall, the amount of free Ca²⁺ cross-linked with alginate in the formed microspheres was in the following order: calcium acetate > calcium chloride + calcium acetate > calcium chloride + calcium gluconate; calcium chloride + calcium lactate > calcium chloride. In microencapsulation by emulsification, the mean size of the microspheres produced can be modified by varying the tack, pH and Ca²⁺ content of the cross-linking solution and through the use of a combination of calcium salts. The shape of the microspheres produced was, nonetheless, unaffected by the physicochemical properties of the cross-linking solution.