The effect of arginine vasopressin, lysine vasopressin or oxytocin on ADP or arginine vasopressin-induced Ca2+ increase in human platelets.

Oxytocin (OT), lysine vasopressin (L8VP), arginine vasopressin (A8VP) or arginine vasotocin (A8VT) are found in plasma from several species and stimulate various cell types by activation of the polyphosphoinositide metabolism and mobilization of intracellular calcium. We therefore studied the effects of the nonapeptides OT, A8VT, L8VP or A8VP on cytoplasmic calcium (Ca2+) and the effect of the nonapeptides on A8VP-induced Ca2+ mobilization. The preincubation of platelets with 'physiological' concentrations of A8VP (2-4 pmol l-1) did not enhance the intracellular calcium increase caused by the agonists used. However, the ADP-induced calcium increase was enhanced by prior addition of subthreshold concentrations of A8VP (less than 1 nmol l-1) to the platelet suspension (by 10%, P = 0.027, n = 12). Neither OT nor A8VT in concentrations from 10(-5) mumol l-1 to 1 mumol l-1 increased the cytoplasmic calcium concentration. We found that both OT and A8VT blocked the effect of subsequent exposure to A8VP. ADP (0.4 mumol l-1) did not block the effect of A8VP.     

Role of G-proteins in the regulation of organic osmolyte efflux from isolated rat renal inner medullary collecting duct cells

 Hypotonic shock (change of osmolality from 600 mosmol to 300 mosmol by lowering NaCl concentration) increases the release of organic osmolytes from isolated inner medullary collecting duct (IMCD) cells in the following sequence: taurine > betaine > sorbitol > myo-inositol > glycerophosphorylcholine (GPC). The role of G-proteins in regulating the hypotonicity-induced efflux was analysed by exposing cells to various concentrations of a G-protein inhibitor, pertussis toxin (PTX; 20–200 ng/ml), and a G_iα-protein stimulator, mastoparan (10–50 μM). PTX diminished the hypotonic release of sorbitol and betaine by 43.2±9.5% and 32.2±7.8% (n = 5), respectively. Efflux of GPC, myo-inositol and taurine was not significantly altered. Mastoparan (10 μM) increased osmolyte release under isotonic conditions such that release of betaine was increased 3.8-fold and that of sorbitol 2.1-fold, while GPC, myo-inositol and taurine effluxes were only slightly augmented. Under hypotonic conditions, mastoparan stimulated betaine release (1.86±0.2-fold, n = 5) but not that of sorbitol. As tested in connection with sorbitol and betaine release, the effect of mastoparan was abolished by PTX, but not the A23187-evoked sorbitol release. Like mastoparan, arachidonic acid increased the release of sorbitol and betaine under isotonic conditions, but under hypotonic conditions it only increased the release of betaine. As to the role of intracellular Ca²⁺, hypotonic shock evoked an intracellular Ca²⁺ peak which could be prevented by PTX. Mastoparan increased intracellular Ca²⁺ under isotonic conditions, whether the extracellular Ca²⁺ concentration was low or high. The results indicate that G-proteins are involved in regulating sorbitol and betaine efflux from IMCD cells. The G-proteins regulating sorbitol release are probably involved in generating the proper intracellular Ca²⁺ signal. Betaine efflux, which is independent of intracellular Ca²⁺, might be regulated by a G-protein-stimulated release of arachidonic acid. Thus, probably several G-proteins are involved in controlling organic osmolyte efflux from IMCD cells. Received: 2 April 1996 / Received after revision: 30 June 1996 / Accepted 25 July 1996     

Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687 mosmol l⁻¹) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced in cells expressing a constitutively active G protein, Rac (RacV₁₂A₃ cell), indicating that Rac acts upstream to caspase-3 activation. The stress-activated protein kinase, p38, was significantly activated by phosphorylation within 30 min after induction of osmotic shrinkage, the phosphorylation being accelerated in fibroblasts overexpressing Rac. Conversely, the activation of the extracellular signal-regulated kinase (Erk1/2) was initially significantly decreased. Subsequent to activation of p38, p53 was activated through serine-15 phosphorylation, and active p53 was translocated from the cytosol to the nucleus. Inhibition of p38 in Rac cells reduced the activation of both p53 and caspase-3. After 60 min in hypertonic medium the rate constants for K⁺ and taurine efflux were increased, particular in Rac cells. We suggest the following sequence of events in the cell shrinkage-induced apoptotic response: cellular shrinkage activates Rac, with activation of p38, followed by phosphorylation and nuclear translocation of p53, resulting in permeability increases and caspase-3 activation.     

Independence of extracellular tortuosity and volume fraction during osmotic challenge in rat neocortex

The structural properties of brain extracellular space (ECS) are summarised by the tortuosity (λ) and the volume fraction (α). To determine if these two parameters were independent, we varied the size of the ECS by changing the NaCl content to alter osmolality of bathing media for rat cortical slices. Values of λ and α were extracted from diffusion measurements using the real-time ionophoretic method with tetramethylammonium (TMA⁺). In normal medium (305 mosmol kg⁻¹), the average value of λ was 1.69 and of α was 0.24. Reducing osmolality to 150 mosmol kg⁻¹, increased λ to 1.86 and decreased α to 0.12. Increasing osmolality to 350 mosmol kg⁻¹, reduced λ to about 1.67 where it remained unchanged even when osmolality increased further to 500 mosmol kg⁻¹. In contrast, α increased steadily to 0.42 as osmolality increased. Comparison with previously published experiments employing 3000 M _r dextran to measure λ, showed the same behaviour as for TMA⁺, including the same constant λ in hypertonic media but with a steeper slope in the hypotonic solutions. These data show that λ and α behave differently as the ECS geometry varies. When α decreases, λ increases but when α increases, λ rapidly attains a constant value. A previous model allowing cellular shape to alter during osmotic challenge can account qualitatively for the plateau behaviour of λ.     

Adenosine inhibition via A1 receptor of N-type Ca2+ current and peptide release from isolated neurohypophysial terminals of the rat

Effects of adenosine on voltage-gated Ca²⁺ channel currents and on arginine vasopressin (AVP) and oxytocin (OT) release from isolated neurohypophysial (NH) terminals of the rat were investigated using perforated-patch clamp recordings and hormone-specific radioimmunoassays. Adenosine, but not adenosine 5'-triphosphate (ATP), dose-dependently and reversibly inhibited the transient component of the whole-terminal Ba²⁺ currents, with an IC₅₀ of 0.875 μ m. Adenosine strongly inhibited, in a dose-dependent manner (IC₅₀= 2.67 μ m ), depolarization-triggered AVP and OT release from isolated NH terminals. Adenosine and the N-type Ca²⁺ channel blocker ω-conotoxin GVIA, but not other Ca²⁺ channel-type antagonists, inhibited the same transient component of the Ba²⁺ current. Other components such as the L-, Q- and R-type channels, however, were insensitive to adenosine. Similarly, only adenosine and ω-conotoxin GVIA were able to inhibit the same component of AVP release. A₁ receptor agonists, but not other purinoceptor-type agonists, inhibited the same transient component of the Ba²⁺ current as adenosine. Furthermore, the A₁ receptor antagonist 8-cyclopentyltheophylline (CPT), but not the A₂ receptor antagonist 3, 7-dimethyl-1-propargylxanthine (DMPGX), reversed inhibition of this current component by adenosine. The inhibition of AVP and OT release also appeared to be via the A₁ receptor, since it was reversed by CPT. We therefore conclude that adenosine, acting via A₁ receptors, specifically blocks the terminal N-type Ca²⁺ channel thus leading to inhibition of the release of both AVP and OT.     

Polyamine Flux in Xenopus Oocytes Through Hemi-Gap Junctional Channels

Diverse polyamine transport systems have been described in different cells, but the molecular entities that mediate polyamine influx and efflux remain incompletely defined. We have previously demonstrated that spermidine efflux from oocytes is a simple electrodiffusive process, inhibitable by external Ca²⁺, consistent with permeation through a membrane cation channel. Hemi-gap junctional channels in Xenopus oocytes are formed from connexin 38 (Cx38), and produce a calcium-sensitive ( I _c) current that is inhibited by external Ca²⁺. Spermidine efflux is also calcium sensitive, and removal of external calcium increases both I _c currents and spermidine efflux in Xenopus oocytes. Injection of Cx38 cRNA or Cx38 antisense oligonucleotides (to increase or decrease, respectively, Cx38 expression) also increases or decreases spermidine efflux in parallel. Spermidine efflux has a large voltage-dependent component, which is abolished with injection of Cx38 antisense oligonucleotides. In addition, spermidine uptake is significantly increased in Cx38 cRNA-injected oocytes in the absence of external calcium. The data indicate that hemi-gap junctional channels provide the Ca²⁺-inhibited pathway for electrodiffusive efflux of polyamines from oocytes, and it is likely that hemi-gap junctional channels provide Ca²⁺ and metabolism-sensitive polyamine permeation pathways in other cells.     

The contribution of intracellular calcium stores to mEPSCs recorded in layer II neurones of rat barrel cortex

Loading slices of rat barrel cortex with 50 μ m BAPTA-AM while recording from pyramidal cells in layer II induces a marked reduction in both the frequency and amplitudes of mEPSCs. These changes are due to a presynaptic action. Blocking the refilling of Ca²⁺ stores with 20 μ m cyclopiazonic acid (CPA), a SERCA pump inhibitor, in conjunction with neuronal depolarisation to activate Ca²⁺ stores, results in a similar reduction of mEPSCs to that observed with BAPTA-AM, indicating that the source for intracellular Ca²⁺ is the endoplasmic reticulum. Block or activation of ryanodine receptors by 20 μ m ryanodine or 10 m m caffeine, respectively, shows that a significant proportion of mEPSCs are caused by Ca²⁺ release from ryanodine stores. Blocking IP₃ receptors with 14 μ m 2-aminoethoxydiphenylborane (2APB) also reduces the frequency and amplitude of mEPSCs, indicating the involvement of IP₃ stores in the generation of mEPSCs. Activation of group I metabotropic receptors with 20 μ m ( RS) -3,5-dihydroxyphenylglycine (DHPG) results in a significant increase in the frequency of mEPSCs, further supporting the role of IP₃ receptors and indicating a role of group I metabotropic receptors in causing transmitter release. Statistical evidence is presented for Ca²⁺-induced Ca²⁺ release (CICR) from ryanodine stores after the spontaneous opening of IP₃ stores.     

Interplay between SERCA and sarcolemmal Ca2+ efflux pathways controls spontaneous release of Ca2+ from the sarcoplasmic reticulum in rat ventricular myocytes

Waves of calcium-induced calcium release occur in a variety of cell types and have been implicated in the origin of cardiac arrhythmias. We have investigated the effects of inhibiting the SR Ca²⁺-ATPase (SERCA) with the reversible inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquinone (TBQ) on the properties of these waves. Cardiac myocytes were voltage clamped at a constant potential between −65 and −40 mV and spontaneous waves evoked by increasing external Ca²⁺ concentration to 4 m m . Application of 100 μ m TBQ decreased the frequency of waves. This was associated with increases of resting [Ca²⁺]_i, the time constant of decay of [Ca²⁺]_i and the integral of the accompanying Na⁺–Ca²⁺ exchange current. There was also a decrease in propagation velocity of the waves. There was an increase of the calculated Ca²⁺ efflux per wave. The SR Ca²⁺ content when a wave was about to propagate decreased to 91.7 ± 3.2%. The period between waves increased in direct proportion to the Ca²⁺ efflux per wave meaning that TBQ had no effect on the Ca²⁺ efflux per unit time. We conclude that (i) decreased wave frequency is not a direct consequence of decreased Ca²⁺ pumping by SERCA between waves but, rather, to more Ca²⁺ loss on each wave; (ii) inhibiting SERCA increases the chance of spontaneous Ca²⁺ release propagating at a given SR content.     

Differences in spike train variability in rat vasopressin and oxytocin neurons and their relationship to synaptic activity

The firing pattern of magnocellular neurosecretory neurons is intimately related to hormone release, but the relative contribution of synaptic versus intrinsic factors to the temporal dispersion of spikes is unknown. In the present study, we examined the firing patterns of vasopressin (VP) and oxytocin (OT) supraoptic neurons in coronal slices from virgin female rats, with and without blockade of inhibitory and excitatory synaptic currents. Inhibitory postsynaptic currents (IPSCs) were twice as prevalent as their excitatory counterparts (EPSCs), and both were more prevalent in OT compared with VP neurons. Oxytocin neurons fired more slowly and irregularly than VP neurons near threshold. Blockade of Cl⁻ currents (including tonic and synaptic currents) with picrotoxin reduced interspike interval (ISI) variability of continuously firing OT and VP neurons without altering input resistance or firing rate. Blockade of EPSCs did not affect firing pattern. Phasic bursting neurons (putative VP neurons) were inconsistently affected by broad synaptic blockade, suggesting that intrinsic factors may dominate the ISI distribution during this mode in the slice. Specific blockade of synaptic IPSCs with gabazine also reduced ISI variability, but only in OT neurons. In all cases, the effect of inhibitory blockade on firing pattern was independent of any consistent change in input resistance or firing rate. Since the great majority of IPSCs are randomly distributed, miniature events (mIPSCs) in the coronal slice, these findings imply that even mIPSCs can impart irregularity to the firing pattern of OT neurons in particular, and could be important in regulating spike patterning in vivo . For example, the increased firing variability that precedes bursting in OT neurons during lactation could be related to significant changes in synaptic activity.     

Components of taurine efflux in rat brain synaptosomes.

The efflux of [³⁵S]taurine from isolated rat brain synaptosomes was studied in a superfusion system. The spontaneous efflux of taurine was slow and could be described as comprising two first-order rate components, of which the slower (t_1/2 = 77.0min) represents release from intrasynaptosomal spaces. Only a high taurine concentration (10 mmol/1) in the medium enhanced the efflux of intrasynaptosomal taurine, which indicates a rather stable intrasynaptosomal taurine compartment. Depolarizing concentrations of potassium ions stimulated taurine efflux and also induced the appearance of a new ‘intermediate’ efflux component. This component was absent when calcium ions were omitted from the medium. It is therefore suggested that the component may originate from the emptying of synaptic vesicles.     

Hypotonicity stimulates translocation of ICln in neonatal rat cardiac myocytes

 Cell volume expansion stimulates the efflux of solutes, including the amino acid taurine, to accomplish a regulatory volume decrease (RVD). One protein that may play a role in taurine efflux is the cytosolic protein ICln. In rat neonatal cardiac myocytes under isotonic conditions, ICln is found predominantly (greater than 90%) in the cytosol. However, after cell volume expansion by exposure to hypotonic medium, ICln rapidly translocates to the particulate fraction (the Triton X-114-insoluble fraction). After 2 min in hypotonic medium the percentage of ICln in the particulate fraction increases to 30%, 46% at 5 min, 40% at 10 min, and 25% at 30 min. The time course of this response is similar to that of hypotonicity-stimulated taurine efflux. Hypotonicity-stimulated taurine efflux as well as ICln translocation parallel the reduction in medium osmolarity. As osmolarity decreases, taurine efflux and ICln movement increase. The movement of ICln from the particulate back to the cytosolic fraction is accelerated when volume-expanded cells are returned to isotonic medium. When ICln is analyzed under non-denaturing conditions, a dimer is detected in the particulate fraction of volume-expanded cells, along with the monomer. This dimer is not detected in the cytosol. Treatment of the particulate fraction from volume-expanded cells with the lyotropic agent KSCN caused release of ICln but not Na-K-ATPase into the soluble fraction, indicating that translocated ICln associates with membranes in the particulate fraction rather than inserting into them. Received: 31 October 1997 / Received after revision and accepted: 23 March 1998     

低渗刺激后大鼠视上核星形胶质细胞释放牛磺酸调节神经元的活动

目的:观察低渗刺激大鼠后,视上核(SON)内星形胶质细胞调节神经元活动的机制。方法:大鼠分为4组:(1)等渗对照组,经尾静脉注入生理盐水;(2)低渗刺激组,经尾静脉注入低渗盐水(0.83%葡萄糖+0.3%NaCl);(3)氟代柠檬酸(胶质细胞代谢抑制剂,fluorocitrate,FCA)+低渗刺激组,经侧脑室注射FCA(1 nmol/μl/只)2 h后,再经尾静脉注入低渗盐水;(4)甘珀酸(缝隙连接通道阻断剂,carbennoxolon,CBX)+低渗刺激组,经侧脑室注射CBX(50μg/只,10μg/lμl)2 h后,再经尾静脉注入低渗盐水。各组动物刺激后90 min,常规固定、取材、下丘脑连续冠状切片。应用抗牛磺酸(taurine,Tau)、抗加压素(vasopressin,VP)、抗甘氨酸受体(glycine recep-tor,GlyR)、抗缝隙连接蛋白(connexin43,Cx43)的抗体进行标记,以及Fos/胶质原纤维酸性蛋白(GFAP)双标记的免疫荧光染色方法,观察其在SON神经元和星形胶质细胞内的表达。结果:与对照组相比,低渗刺激组大鼠,SON内星形胶质细胞的胞体增大、突起变粗;Tau,Cx43和GFAP的平均荧光强度(MFI)增强,神经元上的GlyR表达增强,但VP和Fos的表达减弱。FCA,而不是CBX,明显减少星形胶质细胞的Tau,GFAP,Cx43的表达。FCA和CBX均可抑制神经元上的VP、GlyR、Fos阳性反应。结论:低渗刺激激活SON内星形胶质细胞,被激活的星形胶质细胞经Cx43半通道释放牛磺酸而抑制神经元的活性,减少VP的释放。     

Influence of hypernatraemia and urea excretion on the ability to excrete a maximally hypertonic urine in the rat

Rats normally excrete 20-25 mmol of sodium (Na⁺) + potassium (K⁺) per kilogram per day. To minimize the need for a large water intake, they must excrete urine with a very high electrolyte concentration (tonicity). Our objective was to evaluate two potential factors that could influence the maximum urine tonicity, hypernatraemia and the rate of urea excretion. Balance studies were carried out in vasopressin-treated rats fed a low-electrolyte diet. In the first series, the drinking solution contained an equivalent sodium chloride (NaCl) load at 150 or 600 mmol l⁻¹. In the second series, the maximum urine tonicity was evaluated in rats consuming 600 mmol l⁻¹ NaCl with an 8-fold range of urea excretion. Hypernatraemia (148 ± 1 mmol l⁻¹) developed in all rats that drank 600 mmol l⁻¹ saline. Although the rate of Na⁺+ K⁺ excretion was similar in both saline groups, the maximum urine total cation concentration was significantly higher in the hypernatraemic group (731 ± 31 vs . 412 ± 37 mmol l⁻¹). Only when the rate of excretion of urea was very low, was there a further increase in the maximum urine total cation concentration (1099 ± 118 mmol l⁻¹). Thus hypernatraemia was the most important factor associated with a higher urine tonicity.     

α1-Adrenoceptor-activated cation currents in neurones acutely isolated from rat cardiac parasympathetic ganglia

The noradrenaline (NA)-induced cation current was investigated in neurones freshly isolated from rat cardiac parasympathetic ganglia using the nystatin-perforated patch recording configuration. Under current-clamp conditions, NA depolarized the membrane, eliciting repetitive action potentials. NA evoked an inward cation current under voltage-clamp conditions at a holding potential of −60 mV. The NA-induced current was inhibited by extracellular Ca²⁺ or Mg²⁺, with a half-maximal concentration of 13 μ m for Ca²⁺ and 1.2 m m for Mg²⁺. Cirazoline mimicked the NA response, and prazosin and WB-4101 inhibited the NA-induced current, suggesting the contribution of an α₁-adrenoceptor. The NA-induced current was inhibited by U73122, a phospholipase C (PLC) inhibitor. The membrane-permeable IP₃ receptor blocker xestospongin-C also blocked the NA-induced current. Furthermore, pretreatment with thapsigargin and BAPTA-AM could inhibit the NA response while KN-62, phorbol 12-myristate 13-acetate (PMA) and staurosporine had no effect. These results suggest that NA activates the extracellular Ca²⁺- and Mg²⁺-sensitive cation channels via α₁-adrenoceptors in neurones freshly isolated from rat cardiac parasympathetic ganglia. This activation mechanism also involves phosphoinositide breakdown, release of Ca²⁺ from intracellular Ca²⁺ stores and calmodulin. The cation channels activated by NA may play an important role in neuronal membrane depolarization in rat cardiac ganglia.     

Mechanisms underlying oxytocin-induced excitation of supraoptic neurons: prostaglandin mediation of actin polymerization

In nonneuronal tissues, activation of oxytocin receptors (OTRs), like other Galpha(q/11) type G-protein-coupled receptors (Galpha(q/11)/GPCRs), increase prostaglandin (PG) expression. This is not known for the OTRs expressed by central OT neurons. We examined mechanisms underlying OT's effects on supraoptic nucleus (SON) OT and vasopressin (VP) neurons in hypothalamic slices from lactating rats. OT application (10 pM, 10 min) significantly increased firing rates of OT and VP neurons, both of which expressed OTRs. Indomethacin, an inhibitor of PG synthetases, blocked these increases. OTR (but not a V1 receptor) antagonist blocked OT effects without blocking the excitatory effect of PGE2. Tetanus toxin blocked OT effects on fast synaptic inputs and firing activity of SON neurons but not OT-evoked depolarization, suggesting involvement of both pre- and postsynaptic neurons. Indomethacin also blocked the excitatory effects of phenylephrine, another Galpha(q/11)/GPCR activating agent but not those of PGE2, a non-Galpha(q/11)/GPCR activating agent in the SON. OT or phenylephrine, but not glutamate or KCl, enhanced cyclooxygenase 2 expression at cytosolic loci in SON neurons and nearby astrocytes, as revealed by immunocytochemistry. This OT effect was not blocked by TTX. Western blot analyses showed that OT significantly increased cyclooxygenase 2 but not actin expression. OT promoted the formation of filamentous actin (F-actin) networks at membrane subcortical areas of both OT and VP neurons. Indomethacin blocked enhancement of F-actin networks by OT but not by PGE2. These results indicate that PGs serve as a common mediator of Galpha(q/11)/GPCR-activating agents in neuronal function.     

ATP release from human airway epithelial cells studied using a capillary cell culture system

Epithelial release of adenosine triphosphate (ATP), an important autocrine and paracrine signalling molecule, is acutely mechanosensitive and therefore difficult to study. We describe here a novel preparation that minimizes mechanical and metabolic perturbations, and use it to examine ATP secretion by epithelial cells. The Calu-3 cell line derived from human airway sub-mucosal glands was cultured in a hollow fibre bioreactor on porous capillaries that were perfused by a re-circulating medium pump. Cells became polarized and cultures were stable for > 5 months, as evidenced by microscopy and lactate production (≈250 μg (10⁸ cells)⁻¹ day⁻¹). Elevating apical flow rate 5-fold increased ATP secretion from ≈200 to 6618 fmol min⁻¹. Reducing apical osmolarity by 25–43 % also increased ATP secretion, which then declined spontaneously to a plateau rate that persisted as long as hypotonic perfusion was maintained. Release deactivated rapidly after shear and osmotic stresses were terminated, and was not associated with detectable cell lysis. Lowering apical [Ca²⁺] to increase connexin hemichannel permeability also stimulated ATP release and increased secretion during both hyposmotic and shear stress; however, the connexin 43 blocker flufenamic acid inhibited shear-induced ATP release only in low-Ca²⁺ solution, and therefore another secretory pathway may operate with physiological (i.e. m m ) calcium. Regardless of the mechanism, the present results quantify ATP responses to mechanical and osmotic stimuli and demonstrate the usefulness of capillary cultures for studying epithelial secretion.     

The role of the actin cytoskeleton in oxytocin and vasopressin release from rat supraoptic nucleus neurons

Magnocellular neurons of the supraoptic nucleus (SON) can differentially control peptide release from the somato/dendritic and axon terminal compartment. Dendritic release can be selectively regulated through activation of intracellular calcium stores by calcium mobilizers such as thapsigargin (TG), resulting in preparation (priming) of somato/dendritic peptide pools for subsequent activity-dependent release. As dynamic modulation of the actin cytoskeleton is implicated in secretion from synaptic terminals and from several types of neuroendocrine cells, we studied its involvement in oxytocin and vasopressin release from SON neurons. Confocal image analysis of the somata revealed that the normally continuous cortical band of F-actin is disrupted after high potassium (K⁺, 50 m m ) or TG (200 n m ) stimulation. The functional importance of actin remodelling was studied using cell-permeable actin polymerizing (jasplakinolide, 2 μ m ) or depolymerizing agents (latrunculin B, 5 μ m ) to treat SON and neural lobe (NL) explants in vitro and measure high K⁺-induced oxytocin and vasopressin release. Latrunculin significantly enhanced, and jasplakinolide inhibited, high-K⁺-evoked somato/dendritic peptide release, while release from axon terminals was not altered, suggesting that high-K⁺-evoked release in the SON, but not the NL, requires depolymerization of the actin cytoskeleton. TG-induced priming of somato/dendritic release was also blocked by jasplakinolide and latrunculin, suggesting that priming involves changes in actin remodelling.     

Role of ATP-conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions

It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP-conductive maxi-anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin–luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling-induced ATP release was inhibited by the carboxylate- and stilbene-derivative anion channel blockers, arachidonic acid and Gd³⁺, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch-clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single-channel events with a large unitary conductance (∼390 pS). The channel was selective to anions and showed significant permeability to ATP^4- ( P _ATP/ P _Cl∼ 0.1) and MgATP^2- ( P _ATP/ P _Cl∼ 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi-anion channels.     

Calcium-induced calcium release regulates action potential generation in guinea-pig sympathetic neurones

Experiments were done using guinea-pig sympathetic neurones dissociated from the stellate ganglia to establish whether calcium-induced calcium release (CICR) modulated action potential (AP) generation in mammalian neurones. Using measurements of intracellular calcium ([Ca²⁺]_i) with the Ca²⁺-sensitive dye fluo-3, we demonstrated that 10 m m caffeine activated ryanodine receptors and caused a rise in [Ca²⁺]_i in both Ca²⁺-containing and Ca²⁺-deficient solutions. We also demonstrated that combined treatment with caffeine and 1 μ m thapsigargin or caffeine and 20 μ m ryanodine blocked subsequent caffeine-induced elevations of [Ca²⁺]_i. Treatment with thapsigargin, ryanodine or 200 μ m Cd²⁺ to disrupt CICR decreased the latency to AP generation during 400 ms depolarizing current ramps using the perforated patch whole cell patch clamp in current clamp mode. Treatment with 500 μ m tetraethylammonium also decreased the latency to AP generation during depolarizing current ramps in control cells, but not in cells pretreated with thapsigargin to deplete internal Ca²⁺ stores. In summary, we propose that an outward current, carried at least in part through BK channels, is activated by CICR at membrane voltages approaching the threshold for AP initiation and that this current opposed depolarizing current ramps applied to guinea-pig sympathetic stellate neurones.     

Expression of the sodium–myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells

Myo -inositol is a compatible osmolyte used by cells which are challenged by variations in extracellular osmolarity, as in the renal medulla. In order to accumulate large quantities of this polyol, cells rely on Na⁺-dependent transporters such as SMIT1. We have recently identified a second Na⁺– myo -inositol cotransporter, SMIT2, which presents transport characteristics corresponding to those recently described for the apical membrane of renal proximal tubules. In order to further characterize this transport system, we transfected Madin-Darby canine kidney (MDCK) cells with rabbit SMIT2 cDNA and selected a stable clone with a high expression level. The accumulation of radiolabelled myo -inositol by this cell line is 20-fold larger than that seen in native MDCK cells. The affinity for myo -inositol of MDCK cells transfected with SMIT2 is slightly lower ( K _m= 334 μ m ) than that found in voltage-clamped Xenopus laevis oocytes expressing SMIT2 ( K _m= 120 μ m ). Transport studies performed using semipermeable filters showed complete apical targeting of the SMIT2 transporter. This apical localization of SMIT2 was confirmed by transport studies on purified rabbit renal brush border membrane vesicles (BBMVs). Using a purified antibody against SMIT2, we were also able to detect the SMIT2 protein (molecular mass = 66 kDa) in Western blots of BBMVs purified from SMIT2-transfected MDCK cells. SMIT2 activity was also shown to be stimulated 5-fold when submitted to 24 h hypertonic treatment (+200 mosmol l⁻¹). The SMIT2-MDCK cell line thus appears to be a promising model for studying SMIT2 biochemistry and regulation.