I - Prostaglandin hyperalgesia, a cAMP/Ca dependent process

Prostaglandins stimulate cAMP increase in several biological systems including CNS. The possible participation of a cAMP/Ca²⁺ related mechanism in prostaglandin induced hyperalgesia in the rat paw, as measured by a modification of the Randall-Selitto method was investigated. A serie of agents was administered in the paw in an attempt to change either Ca²⁺ or cyclic AMP concentration at the nociceptive terminations. PGE₂, dibutyryl cyclic AMP, isoprenaline, noradrenaline, adrenaline, Ca²⁺ionophore (A23187), BaCl₂ caused a dose dependent hyperalgesia. The hyperalgesic effect of these substances was enhanced by methyl-xanthines. Cyclic GMP as well as agents which interfere with Ca²⁺ influx(verapamil and lanthanum) were local analgesics in normal and hyperalgesic paws.     

Biphasic effect of extracellular ATP on human and rat airways is due to multiple P2 purinoceptor activation

Background

Extracellular ATP may modulate airway responsiveness. Studies on ATP-induced contraction and [Ca²⁺]_i signalling in airway smooth muscle are rather controversial and discrepancies exist regarding both ATP effects and signalling pathways. We compared the effect of extracellular ATP on rat trachea and extrapulmonary bronchi (EPB) and both human and rat intrapulmonary bronchi (IPB), and investigated the implicated signalling pathways.

Methods

Isometric contraction was measured on rat trachea, EPB and IPB isolated rings and human IPB isolated rings. [Ca²⁺]_i was monitored fluorimetrically using indo 1 in freshly isolated and cultured tracheal myocytes. Statistical comparisons were done with ANOVA or Student''s t tests for quantitative variables and χ² tests for qualitative variables. Results were considered significant at P < 0.05.

Results

In rat airways, extracellular ATP (10^-6–10^-3 M) induced an epithelium-independent and concentration-dependent contraction, which amplitude increased from trachea to IPB. The response was transient and returned to baseline within minutes. Similar responses were obtained with the non-hydrolysable ATP analogous ATP-γ-S. Successive stimulations at 15 min-intervals decreased the contractile response. In human IPB, the contraction was similar to that of rat IPB but the time needed for the return to baseline was longer. In isolated myocytes, ATP induced a concentration-dependent [Ca²⁺]_i response. The contractile response was not reduced by thapsigargin and RB2, a P2Y receptor inhibitor, except in rat and human IPB. By contrast, removal of external Ca²⁺, external Na⁺ and treatment with D600 decreased the ATP-induced response. The contraction induced by α-β-methylene ATP, a P2X agonist, was similar to that induced by ATP, except in IPB where it was lower. Indomethacin and H-89, a PKA inhibitor, delayed the return to baseline in extrapulmonary airways.

Conclusion

Extracellular ATP induces a transient contractile response in human and rat airways, mainly due to P2X receptors and extracellular Ca²⁺ influx in addition with, in IPB, P2Y receptors stimulation and Ca²⁺ release from intracellular Ca²⁺ stores. Extracellular Ca²⁺ influx occurs through L-type voltage-dependent channels activated by external Na⁺ entrance through P2X receptors. The transience of the response cannot be attributed to ATP degradation but to purinoceptor desensitization and, in extrapulmonary airways, prostaglandin-dependent PKA activation.     

Effects of Temperature and Allosteric Modulators on [3H]Nitrendipine Binding: Methods for Detecting Potential Ca2+ Channel Blockers

Abstract

The effects of incubation temperature and allosteric modulators were studied on [³H]nitrendipine binding to guinea-pig cardiac membranes. Incubation temperature only slightly affected the ability of nifedipine and verapamil derivatives to inhibit binding. By contrast, the Ca²⁺ channel blockers d-cis-diltiazem and fostedil (KB-944) stimulated [³H]nitrendipine binding in a temperature-dependent manner (37° > 25° > 4° C). The stimulatory effect of fostedil could be related to a decrease (2.3-fold at 37° C) in the rate of radioligand binding site dissociation, without significant effects on association kinetics. Both fostedil and d-cis-diltiazem caused a shift to the right of the concentration-inhibition curve of tiapamil, a negative allosteric modulator of [³H]nitrendipine binding. Neither compound affected the ability of nifedipine, a competitive antagonist, to inhibit radioligand binding. This selective effect of fostedil or d-cis-diltiazem may be useful for testing whether potential Ca²⁺ channel blockers interact in a competitive as opposed to allosteric manner with the dihydropyridine site. Varying the incubation temperature may also be useful in detecting compounds which act as positive allosteric modulators (stimulators) of dihydropyridine binding.     

The binding of [3H]nitrendipine to receptors for calcium channel antagonists in the heart,cerebral cortex,and ileum of rats

The binding properties of the calcium channel antagonist, [³H]nitrendipine, were investigated in homogenates of the rat cerebral cortex, heart and ileum. The specific component of [³H]nitrendipine binding was consistent with mass-action behavior and was characterized by a high affinity dissociation constant in the range of 0.1 ? 0.3 nM. A variety of other calcium channel antagonists inhibited the binding of [³H]nitrendipine with K_i's that agree generally with the ability of these drugs to block contractions of cardiac and smooth muscle. The inhibition of [³H]nitredipine binding by other dihydropyridines was consistent with competitive antagonism whereas the inhibition caused by verapamil and D600 resembled negative heterotropic cooperativity. Consistent with this latter postulate was the observation that the kinetics of [³H]nitrendipine binding are altered by verapamil, with both the association rate and the dissociation rate being increased. La⁺³ and several divalent cations caused an inhibition of [³H]nitrendipine with the rank order of potency being Cd⁺² > La⁺³ > Ni⁺² > Co⁺² ? Mn⁺² > Mg⁺² ? Ba⁺² > Ca⁺².     

Novel model for “calcium paradox” in sympathetic transmission of smooth muscles: Role of cyclic AMP pathway

It is well established that reduction of Ca²⁺ influx through L-type voltage-dependent Ca²⁺ channel (L-type VDCC), or increase of cytosolic cAMP concentration ([cAMP]c), inhibit contractile activity of smooth muscles in response to transmitters released from sympathetic nerves. Surprisingly, in this work we observed that simultaneous administration of L-type VDCC blocker (verapamil) and [cAMP]c enhancers (rolipram, IBMX and forskolin) potentiated purinergic contractions evoked by electrical field stimulation of rat vas deferens, instead of inhibiting them. These results, including its role in sympathetic transmission, can be considered as a “calcium paradox”. On the other hand, this potentiation was prevented by reduction of [cAMP]c by inhibition of adenylyl cyclase (SQ 22536) or depletion of Ca²⁺ storage of sarco-endoplasmic reticulum by blockade of Ca²⁺ reuptake (thapsigargin). In addition, cytosolic Ca²⁺ concentration ([Ca²⁺]_c) evaluated by fluorescence microscopy in rat adrenal medullary slices was significantly reduced by verapamil or rolipram. In contrast, simultaneous incubation of adrenal slices with these compounds significantly increased [Ca²⁺]_c. This effect was prevented by thapsigargin. Thus, a reduction of [Ca²⁺]_c due to blockade of Ca²⁺ influx through L-type VDCC could stimulate adenylyl cyclase activity increasing [cAMP]c thereby stimulating Ca²⁺ release from endoplasmic reticulum, resulting in augmented transmitter release in sympathetic nerves and contraction.     

Effects of Nitrendipine and Nimodipine on Low-Threshold Ca<Superscript>2+</Superscript> Channels in Thalamic Neurons of the Rat

We compared the effects of two dihydropyridines extensively used in clinical medicine, nimodipine and nitrendipine, on Ca²⁺ channels of two subtypes providing the fast and slow components of low-threshold Ca²⁺ currents in isolated neurons of the nucl. lateralis dorsalis of the thalamus of the rat; a patch-clamp technique in the whole-cell configuration was used. The fast component of the Ca²⁺ current demonstrated a higher sensitivity to nimodipine and nitrendipine (IC₅₀ = 0.6 and 0.1 µM, respectively) than the slow component (IC₅₀ = =1.09 and 0.18 µM, respectively). Maximum decreases in the current amplitude, A _max, caused by nimodipine and nitrendipine were 74 and 94% for the fast component and 55 and 80% for the slow component, respectively. Both tested agents evoked a shift of the inactivation curves of the fast component toward more negative potentials, whereas they did not significantly modify the inactivation characteristics of the slow component. Both dihydropyridines weakly influenced the characteristics of stationary activation of low-threshold Ca²⁺ channels and the blocking intensity demonstrated a voltage dependence.Neirofiziologiya/Neurophysiology, Vol. 37, No. 1, pp. 3–10, January–February, 2005.     

Glutamate Receptor Desensitization Block Potentiates the Stimulated GABA Release Through External Ca2+-Independent Mechanisms from Granule Cells of Olfactory Bulb

Glutamate stimulated release of [³H]GABA was studied, during receptor desensitization block and its modulation by voltage gated Ca²⁺ channels, internal Ca²⁺ mobilization and GABA transport inhibitors from olfactory bulb slices. Under control conditions, glutamate and agonists induced release was strongly inhibited by Mg/0 Ca²⁺ Krebs and Cd²⁺ and partially inhibited by Ni²⁺ and nifedipine. Cyclothiazide, which blocks desensitization of glutamate receptors, potentiated glutamate, kainate, AMPA and quisqualate induced release. This effect was less dependent of entry of external Ca²⁺, but was inhibited by trifluoperazine and thapsigargin, inhibitors of Ca²⁺-calmodulin and endoplasmatic Ca²⁺ ATPase respectively. Nipecotic acid and NO-711, inhibitors of the GABA transporter, were also able to reduce cyclothiazide potentiated release induced by the 4 secretagogues. Under control conditions, glutamate stimulates the release of GABA in cooperation with VDCC. However, during receptor desensitization block, glutamate stimulated GABA release is mainly modulated through mechanisms dependent on internal Ca²⁺ mobilization and reversal of the GABA transporter.     

Endothelin-Mediated Calcium Response and Inositol 1,4,5-Trisphosphate Release in Neuroblastoma-Glioma Hybrid Cells (NG108-15): Cross Talk with ATP and Bradykinin

Abstract: Addition of endothelins (ETs) to neuroblastomaglioma hybrid cells (NG108-15) induced increases in cytosolic free Ca²⁺ ([Ca²⁺]_i) levels of labeled inositol monophosphates and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. The increases in [^Ca2+]_i elicited by the three ETs (ET-1, ET-2, and ET-3) were transient and did not show a sustained phase. Chelating extracellular Ca²⁺ in the medium by adding excess EGTA decreased the ET-mediated Ca²⁺ response by 40-50%. This result indicates that a substantial portion of the increase in [Ca²⁺]_i was due to influx from an extracellular source. However, the increase in [Ca²⁺]_i was not affected by verapamil or nifedipine (10^?5M). A rank order potency of ET-1 ET-2 ET-3 is shown for the stimulated increase in [Ca²⁺]_i, as well as labeled inositol phosphates, in these cells. ATP (10^?4M) and bradykinin (10^?7M) also induced the increases in [Ca²⁺]_i and Ins(1,4,5)P₃ in NG108-15 cells, albeit to a different extent. When compared at 10^?7M, bradykinin elicited a five- to sixfold higher increase in the level of Ins(1,4,5)P₃, but less than a twofold higher increase in [Ca²⁺]_i than those induced by ET-1. Additive increases in both Ins(1,4,5)P₃ and [Ca²⁺]_i were observed when ET-1, ATP, and bradykinin were added to the cells in different combinations, suggesting that each receptor agonist is responsible for the hydrolysis of a pool of polyphosphoinositide within the membrane. ET-1 exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization to the Ca²⁺ response elicited by ATP. On the contrary, ET-1 did not desensitize the response elicited by bradykinin, although bradykinin exhibited complete heterologous desensitization to the response elicited by ET-1. Taken together, these results illustrate that, in NG108-15 cells, a considerable amount of receptor cross talk occurs between ET and other receptors that transmit signals through the polyphosphoinositide pathway.     

Reduction of blood pressure by store‐operated calcium channel blockers

The voltage‐operated Ca²⁺ channels (VOCC), which allow Ca²⁺ influx from the extracellular space, are inhibited by anti‐hypertensive agents such as verapamil and nifedipine. The Ca²⁺ entering from outside into the cell triggers Ca²⁺ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca²⁺ stores in the SR, another type of Ca²⁺ channels in the cell membrane, known as store‐operated Ca²⁺ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd³⁺) sensitive. Both SK and Gd³⁺ have been shown to reduce [Ca²⁺]_i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd³⁺ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd³⁺ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca²⁺]_i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca²⁺]_i. On the other hand, Gd³⁺ inhibited the LPA and Ang II induced change in [Ca²⁺]_i, but had no effect on the ET‐1 and NE induced increase in [Ca²⁺]_i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca²⁺]_i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.     

Stimulation of insulin release by calcium

In the absence of secretagogue, Ca²⁺ (2 to 10 mM) provokes a short-lived release of insulin in the perfused rat pancreas first exposed to EGTA. The secretory response is abolished by verapamil and enhanced by theophylline. These findings afford the first demonstration that Ca²⁺ itself can trigger insulin release.     

Extracellular calcium protects against verapamil-induced metaphase-II arrest and initiation of apoptosis in aged rat eggs

Non-specific L-type calcium channel blockers, such as verapamil (≥50 μM), induce metaphase-II (M-II) arrest and apoptosis in aged rat eggs cultured in Ca²⁺-deficient medium. However, the effects of extracellular Ca²⁺ on verapamil-induced M-II arrest and apoptosis have not yet been reported. We have demonstrated that postovulatory aging induced exit from M-II arrest by extruding a second polar body, a morphological sign of spontaneous egg activation (SEA). Verapamil inhibited SEA and induced egg apoptosis in a dose-dependent manner in Ca²⁺-deficient medium. The initiation of apoptotic features was observed at 50 μM of verapamil. Extracellular Ca²⁺ (1.80 mM) reduced intracellular H₂O₂ level, bax protein expression, caspase-3 activity, DNA fragmentation and protected against 50 μM, but not higher concentrations of ≥100 μM in verapamil-induced egg apoptosis. These results suggest that extracellular Ca²⁺ ions have a role during SEA and protect against verapamil-induced apoptosis in aged rat eggs.     

Modulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by Ca2+ antagonists

Summary The purpose of this study was to examine the effect of three classes of Ca²⁺ antagonists, diltiazem, verapamil and nifedipine on Na⁺-Ca²⁺ exchange mechanism in the sarcolemmal vesicles isolated from canine heart. Na⁺-Ca²⁺ exchange and Ca²⁺ pump (ATP-dependent Ca²⁺ uptake) activities were assessed using the Millipore filtration technique. sarcolemmal vesicles used in this study are estimated to consist of several subpopulations wherein 23% are inside-out and 55% are right side-out sealed vesicles in orientation. The affect of each Ca²⁺ antagonist on the Na⁺-dependent Ca²⁺ uptake was studied in the total population of sarcolemmal vesicles, in which none of the agents depressed the initial rate of Ca²⁺ uptake until concentrations of 10 M were incubated in the incubation medium. However, when sarcolemmal vesicles were preloaded with Ca²⁺ via ATP-dependent Ca²⁺ uptake, cellular Ca²⁺ influx was depressed only by verapamil (28%) at 1 M in the efflux medium with 8 mM Na⁺. Furthermore, inhibition of Ca²⁺ efflux by verapamil was more pronounced in the presence of 16 mM Na⁺ in the efflux medium. The order of inhibition was; verapamil > diltiazem > nifedipine. These results indicate that same forms of Ca²⁺-antagonist drugs may affect the Na⁺-Ca²⁺ exchange mechanism in the cardiac sarcolemmal vesicles and therefore we suggest this site of action may contribute to their effects on the myocardium.     

Post-stimulation retrieval of luminal surface membrane in parotid acinar cells is calcium-dependent

The Ca²⁺ dependence of surface membrane retrieval (i.e., the process by which the excess surface membrane resulting from exocytosis is recycled to the cytoplasm of secretory cells) has been investigated in rat parotid tissue lobules first incubated for 40 min in the presence of a secretagogue drug (the β-adrenergic agonist isoprenaline) and then in the presence of the β-blocker, 1-propranolol, up to 4 h. The dynamics of the luminal surface membrane was monitored by measuring, in ultrathin sections, the length of the luminal profile of all examined acinar cells abutting to a lumen before and immediately at the end of the stimulation, as well as at various times thereafter. Such a profile doubled during isoprenaline stimulation, concomitantly with the discharge of most secretion granules. After the stimulation was blocked, the luminal profile decreased to reach values even lower than those observed in unstimulated cells. The kinetics of this reduction was apparently first-order, both in the presence and in the absence of extracellular Ca²⁺. However, its rate differed appreciably in these two situations: it was relatively fast (apparent ) in lobules incubated in complete medium (Ca²⁺ concentration, 2 mM), and much slower (apparent ) in lobules incubated in a Ca²⁺-free medium containing 1 mM EGTA. The slowing down of the membrane retrieval occurring in Ca²⁺-free conditions was rapidly reversed by reintroduction of Ca²⁺ into the medium. These findings indicate that the retrieval of the luminal surface membrane in parotid acinar cells is Ca²⁺-dependent.     

Differential Contribution of L-, N-, and P/Q-type Calcium Channels to [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> Changes Evoked by Kainate in Hippocampal Neurons

We investigated the contribution of L-, N- and P/Q-type Ca²⁺ channels to the [Ca²⁺]_i changes, evoked by kainate, in the cell bodies of hippocampal neurons, using a pharmacological approach and Ca²⁺ imaging. Selective Ca²⁺ channel blockers, namely nitrendipine, ω-Conotoxin GVIA (ω-GVIA) and ω-Agatoxin IVA (ω-AgaIVA) were used. The [Ca²⁺]_i changes evoked by kainate presented a high variability, and were abolished by NBQX, a AMPA/kainate receptor antagonist, but the N-methyl-d-aspartate (NMDA) receptor antagonist, D-AP5, was without effect. Each Ca²⁺ channel blocker caused differential inhibitory effects on [Ca²⁺]_i responses evoked by kainate. We grouped the neurons for each blocker in three subpopulations: (1) neurons with responses below 60% of the control; (2) neurons with responses between 60% and 90% of the control, and (3) neurons with responses above 90% of the control. The inhibition caused by nitrendipine was higher than the inhibition caused by ω-GVIA or ω-AgaIVA. Thus, in the presence of nitrendipine, the percentage of cells with responses below 60% of the control was 41%, whereas in the case of ω-GVIA or ω-AgaIVA the values were 9 or 17%, respectively. The results indicate that hippocampal neurons differ in what concerns their L-, N- and P/Q- type Ca²⁺ channels activated by stimulation of the AMPA/kainate receptors. Special issue article in honor of Dr. Ricardo Tapia.     

Gonadotropin releasing hormone stimulation of pituitary cell 3′–5′ cyclic AMP in a carp(Cyprinus carpio) is dependent on extracellular calcium

Pituitaries were collected from a common carp,yprinss carpi, belonging to vitellogenic phase and cells were disaggregated by using 0.3% collagenase and 0.05% tsypsin. Enzymatically dispersed cells were incubatedin vitro in Ca²⁺-free medium to observe the effect ofCanna punctatus GnRH (cGnRH) and Ca²⁺ on pituitary cell cAMP accumulation. Addition of cGnRH (20 Big) to pituitary cell incubation (6 × 10⁴ cells/well) containing 4 mM theophylline, a phosphodiesterase inhibitor, caused two-fold increase of cAMP accumulation in comparison to control, Addition of Ca²⁺ (2 mM) to cGnRH further augmented cAMP accumulation, i.e., four-fold as compared to control. Increasing concentrations of cGnRH in the presence of Ca²⁺ resulted in a dose-dependent increase in cAMP accumulation. To examine the specificity of Ca²⁺ augmentory effect on cGnRH-stimulated pituitary cell cAMP accumulation, a specific Ca²⁺-channel blocker, verapamil was used, At 3 μM dose verapamil completely waived Ca²⁺-augmentation of cGnRH stimulatory effect on cAMP. Interestingly, verapamil also significantly inhibited cGnRH stimulation of cAMP in the Ca²⁺-free medium. Extent of Ca²⁺ plus cGnRH stimulatory effect on cAMP was further increased by the addition of 25 pmol of calmodulin, a Ca²⁺-carrier protein, Addition of verapamil to this system strongly inhibited Ca²⁺ and ealmodulin augnientory effect on cGnRH. Reduced level of cAMP in the pituitary cell due to verapamil was even lower than that of cGnRH plus ealmodulin incubation. Data indicates a contamination of Ca²⁺ in an apparently Ca²⁺-free medium, Results suggest that in lower vertebrate, i.e., fish, GnRH stimulation of pituitary cell cAMP is dependent on extracellulnr Ca²⁺ and incubation of pituitary cell in Ca²⁺-free medium is truly not free of Ca²⁺.     

Induction of contracture and extracellular Ca<Superscript>2+</Superscript> influx in cardiac muscle by sanguinarine: a study on cardiotoxicity of sanguinarine

Summary In this study, the toxic effect of sanguinarine (SANG) on heart was studied with isolated cardiac muscle strip isolated from Wistar rat. SANG induced positive inotropic action followed by contracture on the left ventricle and both atria strips. In addition, SANG dose-dependently inhibited spontaneous beat of the right atrium. SANG-induced contracture was completely suppressed by pretreatment with La³⁺ or in a Ca²⁺ free Tyrode solution containing 2.5 mM EGTA. Incubating isolated cardiomyocytes with SANG enhanced the ⁴⁵Ca²⁺ influx, which could be inhibited by pretreatment with La³⁺. However, the SANG-induced ⁴⁵Ca²⁺ influx could not be inhibited by pretreatment with other Ca²⁺ channel blockers, such as nifedipine, verapamil, diltiazem, nickel and manganese, and amiloride. Although antioxidants can inhibit the SANG-induced lipid peroxidation, they could not prevent the SANG-induced contracture. N-acetylcysteine and dithiothreitol, the sulfhydryl reducing agents, were shown to be effective in preventing the SANG-induced contracture. These data suggested that the SANG-induced contracture is caused by the influx of extracellular Ca²⁺ through a La³⁺-sensitive Ca²⁺ channel.     

Cytosolic calcium regulates a potassium current in corn (Zea mays) protoplasts

Summary The voltage- and time-dependent K⁺ current,I _K ⁺ _out , elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La³⁺) to the extracellular medium. These results suggested that the influx of external Ca²⁺ was necessary for K⁺ current activation. The IC₅₀, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca²⁺ actuated the K⁺ current, we altered either the Ca²⁺ buffering capacity or the free Ca²⁺ concentration of the intracellular medium (pipette filling solution). By these means,I _K ⁺ _out could be varied over a 10-fold range. Increasing the free Ca²⁺ concentration from 40 to 400nm also shifted the activation of the K⁺ current toward more negative potentials. Maintaining cytoplasmic Ca²⁺ at 500nm with 40nm EGTA resulted in a more rapid activation of the K⁺ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca²⁺ on depolarization. Increasing intracellular Ca²⁺ to 500nm or 1 m also led to inactivation of the K⁺ current within a few minutes. It is concluded thatI _K ⁺ _out is regulated by cytosolic Ca²⁺, which is in turn controlled by Ca²⁺ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

Calcium Antagonists Modulate 3H-Purine Release from Rat Striatum Glial Cultures Via Vscc-Dependent and Independent Mechanisms

Abstract

Electrical stimulation increased both ³H-purine release and ⁴⁵Ca²⁺ influx in rat primary cultures of astrocytes. Nitrendipine and w-conotoxin reduced the first event being the ion fluxes unaffected. Only when the outward K⁺-currents were impared, 90nM nitrendipine decreased the electrical evoked glial cell inward ⁴⁵Ca²⁺ fluxes too.     

Effect of calcium on growth of submerged <Emphasis Type="Italic">Terfezia boudieri</Emphasis> mycelium

The effect of Ca²⁺ on mycelial growth in Terfezia boudieri was studied. Terfezia boudieri Chatin (Ascomycotinae) occurs in mycorrhizal association with Helianthemum shrubs in deserts with calcareous soils. External Ca²⁺ stimulated mycelial growth in both liquid media and solidified substrates. The response to Ca²⁺ was very faint in well-aerated culture but pronounced in mycelia immersed in the medium, indicating dependence on mycelial aeration. 2,4-Dinitrophenol (DNP), an inhibitor of oxidative phosphorylation, and succinate, a potential cytoplasm acidifier, inhibited mycelial growth but enhanced the stimulatory effect of Ca²⁺. This effect was reduced by the Ca²⁺ channel blocker verapamil.