Presynaptic functional trkB receptors mediate the release of excitatory neurotransmitters from primary afferent terminals in lamina II (substantia gelatinosa) of postnatal rat spinal cord

A subset of primary sensory neurons produces BDNF, which is implicated in control of nociceptive neurotransmission. We previously localized full-length trkB receptors on their terminals within lamina II. To functionally study these receptors, we here employed patch-clamp recordings, calcium imaging and immunocytochemistry on slices from 8-12 days post-natal rats. In this preparation, BDNF (100-500 ng/mL) enhances the release of sensory neurotransmitters (glutamate, substance P, CGRP) in lamina II by acting on trkB receptors expressed by primary afferent fibers of the peptidergic nociceptive type (PN-PAFs). Effect was blocked by trk antagonist K252a or anti-trkB antibody clone 47. A pre-synaptic mechanism was demonstrated after (i) patch-clamp recordings where the neurotrophin induced a significant increase in frequency, but not amplitude, of AMPA-mediated mEPSCs, (ii) real time calcium imaging, where sustained application of BDNF evoked an intense response in up to 57% lamina II neurons with a significant frequency rise. Antagonists of ionotropic glutamate receptors and NK(1) receptors completely inhibited the calcium response to BDNF. Reduction of CGRP (a specific marker of PN-PAFs) and substance P content in dorsal horn following BDNF preincubation, and analysis of the calcium response after depletion with capsaicin, confirmed that the neurotrophin presynaptically enhanced neurotransmitter release from PN-PAFs. This is the first demonstration that trkB receptors expressed by PN-PAF terminals in lamina II are functional during postnatal development. Implications of this finding are discussed considering that BDNF can be released by these same terminals and microglia, a fraction of which (as shown here) contains BDNF also in unactivated state.     

TRPV5 and TRPV6 calcium channels in human T cells

The recent cloning of the special calcium channels TRPV5 and TRPV6 (transient receptor potential vanilloid channels) has provided a molecular basis for studying previously unidentified calcium influx channels in electrically nonexcitable cells. In the present work using RT-PCR, we obtained the endogenous expression of mRNAs of genes trpv5 and trpv6 in lymphoblast leukemia Jurkat cells and in normal human T lymphocytes. Additionally, by immunoblotting, the presence of the channel-forming TRPV5 proteins has been shown both in the total lysate and in crude membrane fractions from Jurkat cells and normal T lymphocytes. The use of immunoprecipitation revealed TRPV6 proteins in Jurkat cells, whereas in normal T lymphocytes, this protein was not detected. The expression pattern and the selective Ca²⁺ permeation properties of TRPV5 and TRPV6 channels indicate the important role of these channels in Ca²⁺ homeostasis, as well as most likely in malignant transformation of blood cells.     

Studies on a PMCA-like protein in the outer mantle epithelium of <Emphasis Type="Italic">Anodonta cygnea</Emphasis>: insights on calcium transcellular dynamics

Early studies on the outer mantle epithelium (OME) cells of the freshwater bivalve Anodonta cygnea (Linnaeus, 1758) revealed high ionic calcium concentrations by electrophysiological methods and subsequently a high tendency to reach an intracellular toxic condition. This toxicity could be neutralized by specific mechanisms in the cytosol of OME cells of A. cygnea. The present immunocytochemistry studies of OME cells by light and transmission electron microscopy (TEM) clearly showed a positive reaction of an antibody directed against the human plasma membrane Ca²⁺-ATPase 1 (PMCA-1) in the cytoplasm of OME cells. Also, western blot analysis of different fractions of OME cells with anti human PMCA-1 and C28R2 antibodies confirmed the presence of a PMCA-like protein with an unusual topographical localization and a molecular weight of only 70–80 kDa. These results lead us to speculate that this PMCA-like protein is distributed either in the plasma membrane or in the entire cytosol, where it eventually regulates intracellular calcium levels. Interestingly, the antibody reactions showed seasonal variations, being highest in OME samples prepared during summer when A. cygnea live under natural acidosis and absent in samples taken in winter conditions, which is in accordance with the seasonal variation of shell calcification rates. During winter, PMCA-1 antibody reaction was also detected in OME cells of animals kept only under experimentally induced acidosis conditions. Therefore, we assume that a functional role for this PMCA-like protein in the intracellular calcium regulation of OME cells during the mineralization of the shells of A. cygnea can be speculated.     

Osthole and imperatorin, the active constituents of Cnidium monnieri (L.) Cusson, facilitate glutamate release from rat hippocampal nerve terminals

We examined the effects of osthole and imperatorin, two active compounds of Cnidium monnieri (L.) Cusson, on the release of glutamate from rat hippocampal synaptosomes and investigated the possible mechanism. The results showed that osthole or imperatorin significantly facilitated 4-aminopridine (4-AP)-evoked glutamate release in a concentration-dependent manner. The facilitatory action of osthole or imperatorin was blocked by the vesicular transporter inhibitor bafilomycin A1, not by the glutamate transporter inhibitor l-transpyrrolidine-2,4-dicarboxylic acid (l-trans-PDC), indicating that the release facilitation by osthole or imperatorin results from a enhancement of vesicular exocytosis and not from an increase of Ca²⁺-independent efflux via glutamate transporter. Examination of the effect of osthole and imperatorin on cytosolic [Ca²⁺] revealed that the facilitation of glutamate release could be attributed to an increase in voltage-dependent Ca²⁺ influx. Consistent with this, ω-conotoxin MVIIC, a wide-spectrum blocker of the N- and P/Q-type Ca²⁺ channels, significantly suppressed the osthole or imperatorin-mediated facilitation of glutamate release, but intracellular Ca²⁺ release inhibitor dantrolene had no effect. Osthole or imperatorin did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization; thus, the facilitation of 4-AP-evoked Ca²⁺ influx and glutamate release produced by osthole or imperatorin was not due to it decreasing synaptosomal excitability. In addition, osthole or imperatorin-mediated inhibition of 4-AP-evoked release was prevented by protein kinase C (PKC) inhibitors. Furthermore, osthole or imperatorin increased 4-AP-induced phosphorylation of PKC. Together, these results suggest that osthole or imperatorin effects a facilitation of glutamate release from nerve terminals by positively modulating N-and P/Q-type Ca²⁺ channel activation through a signaling cascade involving PKC.     

Changes in the level of cytosolic calcium, nitric oxide and nitric oxide synthase activity during platelet aggregation: an in vitro study in platelets from normal subjects and those with cirrhosis

Variceal bleeding due to abnormal platelet function is a well-known complication of cirrhosis. Nitric oxide-related stress has been implicated in the pathogenesis of liver cirrhosis.In the present investigation,we evaluated the level of platelet aggregation and concomitant changes in the level of platelet cytosolic calcium (Ca2+), nitric oxide (NO) and NO synthase (NOS) activity in liver cirrhosis.The aim of the present study was to investigate whether the production of NO by NOS and level of cytosolic Ca2+ influence the aggregation of platelets in patients with cirrhosis of the liver.Agonist-induced aggregation and the simultaneous changes in the level of cytosolic Ca2+, NO and NOS were monitored in platelets of patients with cirrhosis.Platelet aggregation was also measured in the presence of the eNOS inhibitor,diphenylene iodinium chloride (DIC).The level of agonist-induced platelet aggregation was significantly low in the platelets of patients with cirrhosis compared with that in platelets from normal subjects.During the course of platelet aggregation,concomitant elevation in the level of cytosolic Ca2+ was observed in normal samples,whereas the elevation was not significant in platelets of patients with cirrhosis.A parallel increase was observed in the levels of NO and NOS activity.In the presence of the eNOS inhibitor,platelet aggregation was enhanced and accompanied by an elevated calcium level.The inhibition of platelet aggregation in liver cirrhosis might be partly due to greater NO formation by eNOS.Defective Ca2+ release from the internal stores to the cytosol may account for inhibition of aggregation of platelets in cirrhosis.The NO-related defective aggregation of platelets in patients with cirrhosis found in our study is of clinical importance,and the underlying mechanism of such changes suggests a possible therapeutic strategy with cell-specific NO blockers.     

Store-operated Ca2+ entry in astrocytes: different spatial arrangement of endoplasmic reticulum explains functional diversity in vitro and in situ

Ca(2+) signaling is the astrocyte form of excitability and the endoplasmic reticulum (ER) plays an important role as an intracellular Ca(2+) store. Since the subcellular distribution of the ER influences Ca(2+) signaling, we compared the arrangement of ER in astrocytes of hippocampus tissue and astrocytes in cell culture by electron microscopy. While the ER was usually located in close apposition to the plasma membrane in astrocytes in situ, the ER in cultured astrocytes was close to the nuclear membrane. Activation of metabotropic receptors linked to release of Ca(2+) from ER stores triggered distinct responses in cultured and in situ astrocytes. In culture, Ca(2+) signals were commonly first recorded close to the nucleus and with a delay at peripheral regions of the cells. Store-operated Ca(2+) entry (SOC) as a route to refill the Ca(2+) stores could be easily identified in cultured astrocytes as the Zn(2+)-sensitive component of the Ca(2+) signal. In contrast, such a Zn(2+)-sensitive component was not recorded in astrocytes from hippocampal slices despite of evidence for SOC. Our data indicate that both, astrocytes in situ and in vitro express SOC necessary to refill stores, but that a SOC-related signal is not recorded in the cytoplasm of astrocytes in situ since the stores are close to the plasma membrane and the refill does not affect cytoplasmic Ca(2+) levels.     

Dual regulation of the cardiac L-type calcium channel in L6 cells by protein kinase C

The role of protein kinase C (PKC) in the regulation of cardiac L-type Ca²⁺ channel activity (LCC) was investigated in L6 rat neonatal myoblasts. Depolarization of fura-2 loaded cells with 140 mM KCl activated a Ba²⁺ influx pathway that was blocked by nifedipine and stimulated by (−) Bay K 8644. At least two splice variants of the α_1C subunit of the cardiac LCC were identified by PCR; the α_1S subunit of the skeletal muscle LCC was not detected. Peptides that specifically inhibit translocation of the novel, Ca²⁺-independent δ and PKC isozymes reduced Ba²⁺ influx by 27% and 19%, respectively, whereas a corresponding peptide directed against translocation of classical PKC α had no effect. Ingenol 3,20-dibenzoate, an agent reported to selectively activate novel PKCs, increased Ba²⁺ uptake by 31% while ethanol, a PKC agonist, enhanced uptake by 38%. In contrast, selective activation of classical PKCs with thymeleatoxin or an agonist peptide reduced Ba²⁺ influx by 23–33%. Ba²⁺ influx was reduced by 30–40% when cells were treated with either a PKC inhibitor (Gö 6983, bisindolylmaleimide) or the PKC activator phorbol-12-myristate-13-acetate. We propose that novel, Ca²⁺-insensitive PKC(s) enhance cardiac Ca²⁺ channel activity in L6 cells under basal conditions while activation of the classical, Ca²⁺-sensitive PKC(s) inhibits channel activity. These findings provide the first evidence that different PKC isozymes exert class-specific opposing effects on cardiac L-type Ca²⁺ channel activity in L6 myoblasts.     

Magnesium Supplementation did not Affect the Increasing Bone Zinc Concentration in Rats Given Excess Calcium as Carbonate

We investigated the effect of magnesium supplementation on zinc distribution in rats given excess calcium as carbonate. Rats were given a control diet (5 g/kg calcium and 0.5 g/kg magnesium), a high calcium diet (HC, 25 g/kg calcium and 0.5 g/kg magnesium) or the high calcium diet supplied with magnesium (HCM, 25 g/kg calcium and 2.5 g/kg magnesium) for 4 weeks. Calcium carbonate and magnesium oxide were used for increasing these mineral concentrations in diets. Although feed intake did not differ among the groups, the excess calcium suppressed feed efficiency, irrespective of dietary magnesium concentration. Femoral magnesium concentration was lower in the HC group than in the control and the HCM groups. Femoral zinc concentration was higher in the HC group and the HCM group than in the control group. The zinc concentration in the kidney was lower in the HC group and the HCM group than in the control group. The excess calcium did not affect zinc concentration in plasma and other tissues such as the liver, testis, and spleen, irrespective of dietary magnesium. These results suggest that the increasing bone zinc and the decreasing renal zinc do not result from magnesium insufficiency in rats given excess calcium as carbonate.     

Myocardial mechanical dysfunction and calcium overload following rewarming from experimental hypothermia in vivo

Rewarming patients from accidental hypothermia are regularly complicated with cardiovascular instability ranging from minor depression of cardiac output to fatal circulatory collapse also termed “rewarming shock”. Since altered Ca²⁺ handling may play a role in hypothermia-induced heart failure, we studied changes in Ca²⁺ homeostasis in in situ hearts following hypothermia and rewarming. A rat model designed for studies of the intact heart in a non-arrested state during hypothermia and rewarming was used. Rats were core cooled to 15 °C, maintained at 15 °C for 4 h and thereafter rewarmed. As time-matched controls, one group of animals was kept at 37 °C for 5 h. Total intracellular myocardial Ca²⁺ content ([Ca²⁺]_i) was measured using ⁴⁵Ca²⁺. Following rewarming we found a significant reduction of stroke volume and cardiac output compared to prehypothermic control values as well as to time-matched controls. Likewise, we found that hypothermia and rewarming resulted in a more than six-fold increase in [Ca²⁺]_i to 3.01 ± 0.43 μmol/g dry weight compared to 0.44 ± 0.05 μmol/g dry weight in normothemia control. These findings indicate that hypothermia-induced alterations in the Ca²⁺-handling result in Ca²⁺ overload during hypothermia, which may contribute to myocardial failure during and after rewarming.