Homologous and heterologous desensitisation of somatostatin-induced increases in intracellular Ca and inositol 1,4,5-trisphosphate in CHO-K1 cells expressing human recombinant somatostatin sst5 receptors

The mechanisms responsible for somatostatin (SRIF)-induced increases in intracellular Ca²⁺ concentration ([Ca²⁺]_i) and subsequent desensitisation were studied in CHO-K1 cells expressing human sst₅ receptors (CHOsst₅ cells). To study the nature of the desensitisation, interactions with uridine triphosphate (UTP) were examined. SRIF (pEC₅₀ 7.10) and UTP (pEC₅₀ 5.14) caused concentration-dependent increases in [Ca²⁺]_i but the SRIF maximum was about 40% of that to UTP. SRIF-, but not UTP-, induced increases in [Ca²⁺]_i were transient and abolished by pertussis toxin. SRIF and UTP caused sustained increases in Ins(1,4,5)P₃ but the SRIF maximum was about 30% of that to UTP. Removal of [Ca²⁺ ]_e attenuated the SRIF-induced peak rise in [Ca²⁺]_i but had no effect on the peak increases in Ins(1,4,5)P₃. UTP-induced increases in [Ca²⁺]_i and Ins(1,4,5)P₃ were attenuated in the absence of [Ca²⁺]_e. Following pre-exposure to SRIF (1 μM) or UTP (100 μM) for 5 min, subsequent SRIF responses were desensitised. Similar results were obtained in the absence of [Ca²⁺]_e. Pre-exposure to SRIF had no effect on subsequent responses to UTP but in the absence of [Ca²⁺]_e, responses to UTP were attenuated. The results suggest that SRIF but not UTP-induced increases in [Ca²⁺]_i in CHOsst₅ cells are mediated by pertussis toxin sensitive G proteins and are caused by an entry of extracellular Ca²⁺ and release from an Ins(1,4,5)P₃ sensitive Ca²⁺ store. Homologous or heterologous desensitisation of agonist-induced increases in [Ca²⁺]_i could be demonstrated in the presence or absence of extracellular Ca²⁺ respectively, and the latter appeared to involve depletion of a common intracellular Ca²⁺ store.     

Force-frequency relationship in intact mammalian ventricular myocardium: physiological and pathophysiological relevance

The force–frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. The FFR in most mammalian ventricular myocardium is positive; that is, an increase in contractile force in association with an increase in the amplitude of Ca²⁺ transients is induced by elevation of the stimulation frequency, which reflects the cardiac contractile reserve. The relationship is different depending on the range of frequency and species of animal. In some species, including rat and mouse, a ‘primary-phase’ negative FFR is induced over the low-frequency range up to approximately 0.5–1 Hz (rat) and 1–2 Hz (mouse). Even in these species, the FFR over the frequency range close to the physiological heart rate is positive and qualitatively similar to that in larger mammalian species, although the positive FFR is less prominent. The integrated dynamic balance of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) is the primary cellular mechanism responsible for the FFR and is determined by sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ flux through the sarcolemma via L-type Ca²⁺ channels and the Na⁺-Ca²⁺ exchanger. Intracellular Na⁺ concentration is also an important factor in [Ca²⁺]_i regulation. In isolated rabbit papillary muscle, over a lower frequency range (<0.5 Hz), an increase in duration rather than amplitude of Ca²⁺ transients appears to be responsible for the increase in contractile force, while over an intermediate frequency range (0.5–2.0 Hz), the amplitude of Ca²⁺ transients correlates well with the increase in contractile force. Over a higher frequency range (>2.5 Hz), the contractile force is dissociated from the amplitude of Ca²⁺ transients probably due to complex cellular mechanisms, including oxygen limitation in the central fibers of isolated muscle preparations, while the amplitude of Ca²⁺ transients increases further with increasing frequency (‘secondary-phase’ negative FFR). Calmodulin (CaM) may contribute to a positive FFR and the frequency-dependent acceleration of relaxation, although the role of calmodulin has not yet been established unequivocally. In failing ventricular myocardium, the positive FFR disappears or is inverted and becomes negative. The activation and overexpression of cardiac sarcoplasmic reticulum Ca²⁺ ATPase (SERCA2a) is able to reverse these abnormalities. Frequency-dependent alterations of systolic and diastolic force in association with those of Ca²⁺ transients and diastolic [Ca²⁺]_i levels are excellent indicators for analysis of cardiac excitation-contraction coupling, and for evaluating the severity of cardiac contractile dysfunction, cardiac reserve capacity and the effectiveness of therapeutic agents in congestive heart failure.     

CGP37157 modulates mitochondrial Ca homeostasis in cultured rat dorsal root ganglion neurons

The effects of 7-chloro-3,5-dihydro-5-phenyl-1H-4,1-benzothiazepine-2-on (CGP37157), an inhibitor of mitochondrial Na⁺/Ca²⁺ exchange, on depolarization-induced intracellular free Ca²⁺ concentration ([Ca²⁺]_i) transients were studied in cultured rat dorsal root ganglion neurons with indo-1-based microfluorimetry. A characteristic plateau in the recovery phase of the [Ca²⁺]_i transient resulted from mitochondrion-mediated [Ca²⁺]_i buffering. It was blocked by metabolic poisons and was not dependent on extracellular Ca²⁺. CGP37157 produced a concentration-dependent decrease in the amplitude of the mitochondrion-mediated plateau phase (IC₅₀=4±1 μM). This decrease in [Ca²⁺]_i was followed by an increase in [Ca²⁺]_i upon removal of the drug, suggesting that Ca²⁺ trapped in the matrix was released when the CGP37157 was removed from the bath. CGP37157 also inhibited depolarization-induced Ca²⁺ influx at the concentrations required to see effects on [Ca²⁺]_i buffering. Thus, CGP37157 inhibits mitochondrial Na⁺/Ca²⁺ exchange and directly inhibits voltage-gated Ca²⁺ channels, suggesting caution in its use to study [Ca²⁺]_i regulation in intact cells.     

Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca level and contraction in vascular smooth muscle

The effects of isoquinoline derivatives, HA1077 (1-[5-isoquinolinesulfonyl]-homopiperazine) and H-7 (1-[5-isoquinoline-sulfonyl]-2-methylpiperazine), on cytosolic Ca²⁺ levels ([Ca²⁺]_i) and muscle tension were examined in vascular smooth muscle of rat aorta. High K⁺ (72.7 mM) and norepinephrine (1 μM) induced a sustained contraction with a sustained increase in [Ca²⁺]_i. HA1077 and H-7 (3–10 μM) inhibited the increse in muscle tension more strongly than the increase in [Ca²⁺]_i. Verapamil (10 μM) completely inhibited the increase in [Ca²⁺]_i and the contraction induced by K⁺ whereas it inhibited the increase in [Ca²⁺]_i more strongly than the contraction due to norepinephrine. The verapamil-insensitive portion of the norepinephrine-induced contraction was inhibited by HA1077 or H-7. In Ca²⁺-free solution, 0.1 μM norepinephrine induced a transient increase in [Ca²⁺]_i and muscle tension. The transient contraction was inhibited by 10 μM HA1077 or 10 μM H-7 without inhibiting the increase in [Ca²⁺]_i. 12-Deoxyphorbol 13-isobutyrate (DPB) (1 μM) caused a sustained contraction, and this contraction was inhibited by HA1077 and H-7 at similar concentrations needed to inhibit the contractions induced by high K⁺ or norepinephrine. In rabbit mesenteric artery permeabilized with Staphylococcus aureus -toxin, 100 μM HA1077 and 100 μM H-7 inhibited the contraction induced by 0.3 μM Ca²⁺. These results suggest that the inhibitory effects of isoquinoline derivatives, HA1077 and H-7, are due to a decrease in [Ca²⁺]_i and in the Ca²⁺ sensitivity of contractile elemenst in vascular smooth muscle.     

Heptachlor epoxide induces a non-capacitative type of Ca2+ entry and immediate early gene expression in mouse hepatoma cells

The effects of the organochlorine (OC) liver tumor promoter heptachlor epoxide (HE) and a related non-tumor promoting OC, delta-hexachlorocyclohexane (δ-HCH), on the dynamics of intracellular calcium (Ca²⁺) were investigated in mouse 1c1c7 hepatoma cells. HE induced a non-capacitative, Ca²⁺ entry-like phenomenon, which was transient and concentration-dependent with 10 and 50 μM HE. The plasma membrane Ca²⁺ channel blocker SKF-96365 antagonized this HE-induced Ca²⁺ entry. δ-HCH failed to induce Ca²⁺ entry, rather it antagonized the HE-induced Ca²⁺ entry. Both HE and δ-HCH induced Ca²⁺ release from endoplasmic reticulum (ER) at treatment concentrations as low as 10 μM; at 50 μM, the former induced 5× as much Ca²⁺ release as the latter. The HE-induced Ca²⁺ release from the ER was antagonized using the IP₃ receptor/channel blocker xestospongin C, suggesting that HE induces ER Ca²⁺ release through the IP₃ receptor/channel pore. These results show that the effect of HE on cellular Ca²⁺ mimics that of mitogens such as epidermal and hepatocyte growth factors. They also provide insight into the similarities and differences between tumorigenic and non-tumorigenic OCs, in terms of the mechanisms and the extent of the [Ca²⁺]_i increased by these agents.     

Mechanisms of pinacidil-induced vasodilatation

The mechanism of the vasodilator effect of pinacidil was examined. Pinacidil (0.1–100 μM) inhibited the increases in cytosolic Ca²⁺ ([Ca²⁺]_i) and muscle tension due to norepinephrine in rat aorta. In contrast, a Ca²⁺ channel blocker, verapamil, inhibited the norepinephrine-stimulated [Ca²⁺]_i more strongly than the contraction. Higher concentrations of pinacidil (3–100 μM) inhibited the verapamil-insensitive portion of the contraction and [Ca²⁺]_i. An inhibitor of ATP-sensitive K⁺ channels, glibenclamide, antagonized the inhibitory effect of low concentrations ( 10 pM) of pinacidol. Pinacidil did not change the contraction induced by Ca²⁺ in vascular smooth muscle permeabilized with Staphylococcus aureus -toxin. Norepinephrine (in the presence of GTP), 12-deoxyphorbol 13-isobutyrate (in the absence of GTP), and treatment with GTP_γS potentiated the contraction of permeabilized smooth muscle induced by the addition of Ca²⁺. Pinacidil (100 μM) inhibited the potentiation due to GTP_γS or noepinephrine but not to phorbol ester. These results suggest that pinacidil has dual effects on vascular smooth muscle contraction. At lower concentrations (>0.1 μM), it decreases [Ca²⁺]_i, possibly by activating ATP-sensitive K+ channels. At higher concentrations (> 3 μM), it may additionally inhibit the receptor-mediated, GTP-binding protein-coupled phosphatidyl inositol turnover.     

Cytosolic Ca transients in endothelium-dependent relaxation of pig coronary artery, and effects of captopril

To determine the mechanism of endothelium-dependent relaxation by bradykinin, we simultaneously measured changes in cytosolic calcium concentrations ([Ca²⁺]_i) and force of fura-2-loaded strips of porcine coronary artery. We also examined effects of captopril, an angiotensin converting enzyme inhibitor, on bradykinin-induced relaxation. Bradykinin, in a concentration-dependent manner (10⁻¹⁰ to 10⁻⁷ M), decreased both [Ca²⁺]_i and force to resting levels, during 10⁻⁵ M prostaglandin F₂-induced contractions, only when endothelium was intact. Treatment with 10⁻⁵ M captopril enhanced the bradykinin-induced decreases in [Ca²⁺]_i and force and shifted the concentration-response bradykinin induced a greater relaxation than that expected from the reduction in [Ca²⁺]_i. Captopril had no effects on the relationship between reduction in [Ca²⁺]_i and relaxation induced by bradykinin. Bradykinin relaxes porcine coronary artery in an endothelium-dependent manner, by decreasing [Ca²⁺]_i and also by controlling the Ca²⁺ sensitivity of the contractile apparatus of smooth muscle. Captopril enhanced the bradykinin-induced relaxation, with no apparent direct effect on Ca²⁺ sensitivity of the contractile apparatus.     

Effects of Ca channel blockers on Ca loading induced by metabolic inhibition and hyperkalemia in cardiomyocytes

The effects of the L-type (nifedipine and verapamil) and the T-type (mibefradil) Ca²⁺ channel blockers on the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by NaCN metabolic inhibition and hyperkalemia were examined in chicken cardiomyocytes using fluorescence imaging with Fura-2. NaCN induced a slow and sustained rise in [Ca²⁺]_i, which was not affected by pretreating the cells for 5 min with nifedipine, verapamil, or mibefradil at 100 nM or 10 μM. Pretreatment of the cells with 10 μM nifedipine, verapamil, or mibefradil for 5 min remarkably inhibited the K⁺-induced increase in [Ca²⁺]_i. These inhibitory effects diminished after 48-h pretreatment with nifedipine or verapamil but not with mibefradil. Ryanodine also induces an increase in [Ca²⁺]_i, and this effect was enhanced by 48-h pretreatment of the cells with 10 μM verapamil but not with 10 μM mibefradil. We conclude that the NaCN-induced increase in [Ca²⁺]_i is independent of the Ca²⁺ influx though the L-type or T-type Ca²⁺ channels. Chronic inhibition of the L-type Ca²⁺ channels but not T-type channels may enhance the ryanodine receptor-mediated Ca²⁺ release, which may be responsible for the development of tolerance to their inhibitory effects on K⁺-induced increase in [Ca²⁺]_i.     

Sarcoplasmic reticulum Ca2+ release channel ryanodine receptor (RyR2) plays a crucial role in aconitine-induced arrhythmias

The present study established a model of RyR₂ knockdown cardiomyocytes and elucidated the role of RyR₂ in aconitine-induced arrhythmia. Cardiomyocytes were obtained from hearts of neonatal Sprague–Dawlay rats. siRNAs were used to down-regulate RyR₂ expression. Reduction of RyR₂ expression was documented by RT-PCR, western blot, and immunofluorescence. Ca²⁺ signals were investigated by measuring the relative intracellular Ca²⁺ concentration, spontaneous Ca²⁺ oscillations, caffeine-induced Ca²⁺ release, and L-type Ca²⁺ currents. In normal cardiomyocytes, steady and periodic spontaneous Ca²⁺ oscillations were observed, and the baseline [Ca²⁺]_i remained at the low level. Exposure to 3 μM aconitine increased the frequency and decreased the amplitude of Ca²⁺ oscillations; the baseline [Ca²⁺]_i and the level of caffeine-induced Ca²⁺ release were increased but the L-type Ca²⁺ currents were inhibited after application of 3 μM aconitine for 5 min. In RyR₂ knockdown cardiomyocytes, the steady and periodic spontaneous Ca²⁺ oscillations almost disappeared, but were re-induced by aconitine without affecting the baseline [Ca²⁺]_i level; the level of caffeine-induced Ca²⁺ release was increased but L-type Ca²⁺ currents were inhibited. Alterations of RyR₂ are important consequences of aconitine-stimulation and activation of RyR₂ appear to have a direct relationship with aconitine-induced arrhythmias. The present study demonstrates a potential method for preventing aconitine-induced arrhythmias by inhibiting Ca²⁺ leakage through the sarcoplasmic reticulum RyR₂ channel.     

Mechanisms of vasoinhibitory effect of cardioprotective agent, CP-060S, in rat aorta

Vasoinhibitory effects of (−)-(S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3,4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin-4-one hydrogen fumarate (CP-060S), a synthesized cardioprotective agent, were examined. In the rat aortic rings, the contractile responses to cumulative application of angiotensin II, [Arg⁸]-vasopressin (vasopressin), or prostaglandin F₂ were inhibited by CP-060S in a concentration-dependent manner. The Ca²⁺-induced contractions in the presence of vasopressin or prostaglandin F₂ were also inhibited by CP-060S in a concentration-dependent manner. The inhibitory effect of 10⁻⁵ M CP-060S on phenylephrine-induced contraction was as potent as that of 10⁻⁶ M nifedipine, and the combined addition of 10⁻⁶ M nifedipine and 10⁻⁵ M CP-060S showed the effect similar to that of 10⁻⁵ M CP-060S alone. In rat aorta loaded with a Ca²⁺ indicator, fura-PE3, 10⁻⁵ M CP-060S completely inhibited the high K⁺-induced increase in cytosolic Ca²⁺ level ([Ca²⁺]_i) and contraction. In contrast, 10⁻⁵ M CP-060S only partially inhibited the increase in [Ca²⁺]_i and contraction due to phenylephrine or prostaglandin F₂. In the presence of 10⁻⁶ M nifedipine, 10⁻⁵ M CP-060S did not inhibit the increase in [Ca²⁺]_i and contraction induced by prostaglandin F₂. In a Ca²⁺-free medium, the phasic increases in contraction and [Ca²⁺]_i induced by phenylephrine were not affected by 10⁻⁵ M CP-060S. These results suggest that the vasoinhibitory effect of CP-060S in rat aortic rings is due mainly to the inhibition of L-type voltage-dependent Ca²⁺-channels.     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes.

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.

Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).

In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.

Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Induction of cerebellar long-term depression requires activation of calcineurin in Purkinje cells

Cerebellar long-term depression (LTD) is an activity-dependent depression of synaptic transmission from parallel fibers to Purkinje cells underlying certain forms of motor learning. LTD is induced by the conjunctive stimulation of parallel fibers and climbing fibers, both of which supply excitatory inputs to Purkinje cells. The conjunctive stimulation induces a large increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in Purkinje cells. Although the increase in [Ca²⁺]_i is essential for LTD induction, the downstream signal transduction mechanism remains elusive. In this study, we show that LTD induction requires the activation of the Ca²⁺/calmodulin-dependent protein phosphatase 2B calcineurin. In acute cerebellar slices of mice, the LTD amplitude was significantly reduced in the presence of calcineurin inhibitors (cyclosporin A or FK506), whereas the basic electrophysiological properties of the parallel fiber-Purkinje cell synaptic transmission remained constant. Furthermore, a calcineurin autoinhibitory peptide perfused into Purkinje cells completely blocked LTD induction. On the other hand, microcystin LR, an inhibitor of protein phosphatase 1 and 2A, did not affect the induction of LTD. These results indicate that calcineurin activation is essential for LTD induction downstream of the conjunctive-stimulation-induced Ca²⁺ signal in Purkinje cells.     

5-Hydroxytryptamine induces transient Ca influx through Ni-insensitive Ca channels in rat vascular smooth muscle cells

The effects of Ni²⁺, a non-selective cation channel inhibitor, on 5-hydroxytryptamine (5-HT)- and angiotensin II (Ang II)-induced intracellular Ca²⁺ dynamics in rat aortic smooth muscle cells were investigated. Ni²⁺ (1 mM) significantly inhibited the transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by Ang II (100 nM) in aortic smooth muscle cells, as measured using fura-2. However, Ni²⁺ did not suppress the transient increase in Ca²⁺ influx induced by 5-HT (10 μM), while significantly suppressed the sustained increase. Ca²⁺ influx evoked by high KCl (80 mM), thapsigargin (TG) (1 μM) or depletion of intracellular Ca²⁺ store was almost completely suppressed by Ni²⁺. Ni²⁺ had no effect on 5-HT-induced inositol triphosphate production and Ca²⁺ release from the intracellular store(s). These results suggest that 5-HT, but not Ang II, induces transient Ca²⁺ influx through Ni²⁺-insensitive Ca²⁺ channels, which are distinguishable from the voltage-dependent or store-operated Ca²⁺ channels.     

Ca dependence of the response of three adenosine type receptors in rat hepatocytes

The effect of three different receptor-specific adenosine agonists on the rate of ureagenesis by isolated rat hepatocytes and the dependence on the external free Ca²⁺ concentration ([Ca²⁺]_e) were investigated. In the presence of high [Ca²⁺]_e all adenosine receptor agonists increased ureagenesis to similar levels. However, with low [Ca²⁺]_e the effects of each agonist varied as follows: (i) the adenosine A₁ receptor agonist, 2-chloro-N⁶-cyclopentyl-adenosine, increased ureagenesis depending partially on [Ca²⁺]_e, (ii) the adenosine receptor A₂ agonist, 2-p-(-2-carboxy-ethyl) phenethylamino-5′-N-ethylcarboxyamido adenosine hydrochloride, increased ureagenesis independently of [Ca²⁺]_e and (iii) in contrast, the adenosine receptor A₃ agonist N⁶-2-(-4-aminophenyl) ethyladenosine, increased ureagenesis only in the presence of high [Ca²⁺]_e. The adenosine receptor A₁ antagonist, 1-allyl-3,7-dimethyl-8-phenyl xanthine, inhibited the effect of the adenosine receptor A₁ agonist on ureagenesis, but not the effect of the adenosine A₂ or A₃ receptor agonists. The adenosine A₂ receptor antagonist, 3,7-dimethyl-1-propargylxanthine, inhibited only the effect of the adenosine A₂ receptor agonist. Thus, in addition to A₁ and A₂ type adenosine receptors, rat hepatocytes possess an A₃-like adenosine receptor which responds to the addition of an adenosine A₃ agonist by accelerating ureagenesis a [Ca²⁺]_e dependent manner. Moreover, it was observed that in the presence of extracellular Ca²⁺ each agonist increased [Ca²⁺]_i and this effect was inhibited by the appropriate specific antagonist.     

The effects of N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA) on the changes in free intracellular calcium and the production of reactive oxygen metabolites in human leukocytes

Human leukocytes were exposed to N-(5-vinyl-1,3-thiazolidin-2-ylidene)phenylamine (5-VTPA), a postulated impurity in the case oils that caused the Spanish Toxic Oil Syndrome in 1981. Changes induced by 5-VTPA alone and together with a chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP), a tumor promoter, phorbol myristate acetate (PMA), or a synthetic diacylglycerol, dioctanoyl-s,n-glycerol (DiC₈) in free intracellular calcium levels ([Ca²⁺]_i) and in the induction of oxidative burst were measured. 5-VTPA elevated dose-dependently [Ca²⁺]_i and induced the production of reactive oxygen metabolites in leukocytes. 5-VTPA also amplified FMLP-induced increase in [Ca²⁺]_i, but was without an effect on FMLP-induced oxidative burst. On the contrary, 5-VTPA amplified dose-dependently PMA- and DiC₈-induced respiratory burst. The present results indicate that 5-VTPA may interfere with transmembrane signalling in human leukocytes. 5-VTPA may elevate [Ca²⁺]_i by acting directly on the membrane, or by acting through Ca²⁺-mobilizing receptors. Moreover, 5-VTPA also clearly amplified responses produced through protein kinase C stimulation. Thus, 5-VTPA may act on human leukocytes by affecting Ca²⁺-metabolism and the activity of protein kinase C.     

The Protective Action of Tetrodotoxin and (±)-Kavain on Anaerobic Glycolysis, ATP Content and Intracellular Na and Ca of Anoxic Brain Vesicles

Because recent reports point to Na⁺ channel blockers as protective agents directed against anoxia-induced neuronal damage including protection of anaerobic glycolysis, the influences of tetrodotoxin (TTX) and (±)-kavain on anoxic rat brain vesicles were investigated with respect to lactate synthesis, vesicular ATP content and cytosolic free Na⁺ and Ca²⁺ ([Na⁺]_i, [Ca²⁺]_i), both of the latter determined fluorometrically employing SBFI and FURA-2, respectively. After anoxia, basal lactate production was increased from 2.9 to 9.8 nmol lactate/min/mg protein. Although lactate synthesis seemed to be stable for at least 45 min of anoxia, as deduced from the linearity of lactate production, the ATP content declined continuously with a half life (τ ) af 14.5 min, indicating that anaerobic glycolysis was insufficient to cover the energy demand of anoxic vesicles. Correspondingly, [Na⁺]_i and [Ca²⁺]_i increased persistently after anoxia by 22.1 mmol/l Na⁺ and 274.9 nmol/l Ca²⁺, determined 6.3 min after onset. An additional stimulation of vesicles with veratridine accelerated the drop of ATP (τ = 5.1 min) and provoked a massive Na⁺ overload, which levelled off to 119 mmol/l Na⁺ within a few minutes. Concomitantly, [Ca²⁺]_i increased linearly with a rate of 355 nmol Ca²⁺/l/min. Despite the massive perturbation of ion homeostasis, lactate production was unaffected during the first 8 min of veratridine stimulation. However, complete inhibition of lactate synthesis took place 30 min after veratridine was added. The Na⁺ channel blockers TTX and (±)-kavain, if applied before anoxia, preserved vesicular ATP content, diminished anoxia-induced increases in [Na⁺]_i and [Ca²⁺]_i and prevented both the veratridine-induced increases of [Na⁺]_i and [Ca²⁺]_i and the inhibition of lactate production. The data indicate a considerable Na⁺ influx via voltage-dependent Na⁺ channels during anoxia, which speeds up the decline in ATP and provokes an increase in [Ca²⁺]_i. A massive Na⁺ and Ca²⁺ overload induced by veratridine failed to influence lactate synthesis directly, but initiated its inhibition. © 1997 Elsevier Science Ltd. All rights reserved.     

4-hydroxy nimesulide effects on mitochondria and HepG2 cells. A comparison with nimesulide

The effects of puerarin on behaviour and brain neuronal activity in animal studies have been described previously. However, molecule mechanisms underlying these effects were poorly understood. Here, we examined the regulation of puerarin on the Ca²⁺ signals in primary rat hippocampal neurons using Fura-2 based calcium imaging techniques. Application of puerarin had no effect on the basal intracellular calcium concentration ([Ca²⁺]_i), but potentiated the KCl-evoked [Ca²⁺]_i transient in 87% of recorded neurons. Dantrolene or ruthenium red, the inhibitors of ryanodine receptors, completely blocked this potentiation induced by puerarin. Moreover, in Ca²⁺-free solution, pre-application of puerarin significantly augmented the elevation of [Ca²⁺]_i evoked by caffeine (3 mM), which is a specific agent to activate the ryanodine receptors. In contrast, nifedipine failed to prevent the potentiation induced by puerarin. Similarly, in the experiments of whole-cell patch-clamp recording, puerarin did not show any effect on calcium currents generated by depolarization pulses. These data demonstrated that the potentiation induced by puerarin was attributed to the facilitation of Ca²⁺-induced Ca²⁺ release (CICR) via ryanodine receptors, rather than extracellular Ca²⁺ influx. Using estrogen receptor antagonist ICI 182780 and tamoxifen, we further demonstrated that the potentiation induced by puerarin was mediated by the estrogen receptor. Furthermore, the membrane-permeant inhibitor of protein kinase A (PKA) H89 completely inhibited this potentiation. However, U-73122, the inhibitor of phospholipase C (PLC) had no effect, indicating that the cyclic AMP/PKA signaling pathway was involved in the activation of CICR by puerarin.     

Inhibition of N-, P/Q- and other types of Ca channels in rat hippocampal nerve terminals by the adenosine A1 receptor

The effects of the adenosine A₁ receptor agonist, N⁶-cyclopentyladenosine (CPA), on both the increase in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and on the release of endogenous glutamate in rat hippocampal synaptosomes were studied. The inhibitory effect of CPA on the increase in [Ca²⁺]_i stimulated with 4-aminopyridine was neutralized by the adenosine A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). The inhibitory effect of CPA was greater in synaptosomes from the CA1 subregion than in whole hippocampal synaptosomes. The inhibitory effects of both CPA and of the Ca²⁺ channel blockers, ω-conotoxin GVIA, ω-conotoxin MVIIC or ω-conotoxin GVIA plus ω-conotoxin MVIIC, were greater than those caused by the Ca²⁺ channel blockers. The release of endogenous glutamate was inhibited by 41% by CPA. The inhibition observed when CPA and ω-conotoxin GVIA or CPA and ω-conotoxin MVIIC were present was also greater than the inhibition by the Ca²⁺ channel blockers alone. The presence of both ω-conotoxin GVIA and ω-conotoxin MVIIC did not completely inhibit the release of glutamate, and CPA significantly enhanced this inhibition. The membrane potential and the accumulation of [

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]tetraphenylphosphonium of polarized or depolarized synaptosomes was not affected by CPA, suggesting that adenosine did not increase potassium conductances. The present results suggest that, in hippocampal glutamatergic nerve terminals, adenosine A₁ receptor activation partly inhibits P/Q- and other non-identified types of Ca²⁺ channels.     

Effects of [Na] and [Ca] on the responses to milrinone in rat cardiac preparations

In the present investigation we have studied the influence of changing the [Ca²⁺] and [Na⁺] on the cardiac responses to milrinone in various preparations of rat heart. Milrinone (5 × 10⁻⁵ to 8 × 10⁻⁴ M) produced a dose-dependent positive chronotropic effect on right atrium and a positive inotropic effect on left atrium and papillary muscle of the rat. A decrease in [Ca²⁺] (from 2.2 to 1.1 mM) or an increase in [Na⁺] (from 120 to 60 mM) increased the milrinone-induced inotropic effect in left atrium and papillary muscle. However, in right atrium the chronotropic effect of milrinone was significantly decreased under these conditions. Opposite changes to milrinone-induced responses were observed when [Ca²⁺] was increased (to 3.3 mM) or when the [Na⁺] was decreased to 60 mM. Nifedipine (3 × 10⁻³ M), a selective Ca²⁺ channel blocker, significantly inhibited the chronotropic response to milrinone in right atrium. However, the inotropic response to milrinone was found to be significantly greater in the presence of nifedipine. A veratridine-induced positive inotropic effect in the left atrium was also significantly increased in the presence of nifedipine. Tetrodotoxin (TTX, 1 × 10⁻⁶ M), a fast sodium channel blocker, significantly reduced the inotropic response to milrinone in left atrium and papillary muscle. A milrinone-induced dose-dependent increase in the baseline tension was observed in the right atrium which was abolished in low [Ca²⁺] and significantly increased in high [Ca²⁺]. Our data suggest the possibility that milrinone increases Ca²⁺ influx in the right atrium to cause the chronotropic effect. Milrinone also may possess an action like veratridine, involving an increased influx of Na⁺ through fast Na⁺ channels in left atrium and papillary muscle, and this action is possibly involved in the positive inotropic effect.     

Cannabinoids produce neuroprotection by reducing intracellular calcium release from ryanodine-sensitive stores

Exogenously administered cannabinoids are neuroprotective in several different cellular and animal models. In the current study, two cannabinoid CB1 receptor ligands (WIN 55,212-2, CP 55,940) markedly reduced hippocampal cell death, in a time-dependent manner, in cultured neurons subjected to high levels of NMDA (15 μM). WIN 55,212-2 was also shown to inhibit the NMDA-induced increase in intracellular calcium concentration ([Ca²⁺]_i) indicated by FURA-2 fluorescence imaging in the same cultured neurons. Changes in [Ca²⁺]_i occurred with similar concentrations (25–100 nM) and in the same time-dependent manner (pre-exposure 1–15 min) as CB1 receptor mediated neuroprotective actions. Both effects were blocked by the CB1 receptor antagonist SR141716A. An underlying mechanism was indicated by the fact that (1) the NMDA-induced increase in [Ca²⁺]_i was inhibited by ryanodine, implicating a ryanodine receptor (RyR) coupled intracellular calcium channel, and (2) the cannabinoid influence involved a reduction in cAMP cAMP-dependent protein kinase (PKA) dependent phosphorylation of the same RyR levels that regulate channel. Moreover the time course of CB1 receptor mediated inhibition of PKA phosphorylation was directly related to effective pre-exposure intervals for cannabinoid neuroprotection. Control studies ruled out the involvement of inositol-trisphosphate (IP3) pathways, enhanced calcium reuptake and voltage sensitive calcium channels in the neuroprotective process. The results suggest that cannabinoids prevent cell death by initiating a time and dose dependent inhibition of adenylyl cyclase, that outlasts direct action at the CB1 receptor and is capable of reducing [Ca²⁺]_i via a cAMP/PKA-dependent process during the neurotoxic event.