New insights into the effect of VMP1 on the treatment of pressure overload-induced pathological cardiac hypertrophy: Involving SERCA-regulated autophagic flux.

Pathological cardiac hypertrophy is an adaptive reaction in response to pressure or volume overload. Autophagy is critical for damage caused by pathological cardiac hypertrophy. Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that is effective in activating autophagy. However, the role of VMP1 in pathological cardiac hypertrophy and its underlying mechanisms remain elusive. This study was designed to explore the potential mechanisms of VMP1 on pressure overload-induced pathological cardiac hypertrophy. In this work, abdominal aorta constriction (AAC) surgery was used to induce pathological cardiac hypertrophy in male C57BL/6 mice. H9C2 cardiomyocytes were treated with phenylephrine stimulation (PE) to induce the hypertrophic response. The in vivo results revealed that mice with AAC surgery caused pathological cardiac hypertrophy as evidenced by improved cardiac function according to multiple echocardiographic parameters. Moreover, elevated VMP1 expression was also observed in mice after AAC surgery. VMP1 knockdown aggravated changes in cardiac structure, cardiac dysfunction, and fibrosis. Meanwhile, VMP1 knockdown suppressed autophagy and endoplasmic reticulum calcium ATPase (SERCA) activity in heart tissues. H9C2 cardiomyocytes with VMP1 overexpression were used to investigate the specific mechanism of VMP1 in pathological cardiac hypertrophy, and VMP1 overexpression increased autophagic flux by upregulating SERCA activity. In conclusion, these findings revealed that VMP1 protected against pressure overload-induced pathological cardiac hypertrophy by inducing SERCA-regulated autophagic flux. Our results provide valuable insights regarding the pathophysiology of pathological cardiac hypertrophy and clues to a novel target for the treatment of pathological cardiac hypertrophy.

Reduced Plasma-Membrane Calcium ATPase Activity and Extracellular Acidification Trigger Presynaptic Homeostatic Potentiation at the Mouse Neuromuscular Junction.

At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to an increase in neurotransmitter release that restores the strength of synaptic transmission following a blockade of nicotinic acetylcholine receptors (nAChRs). Mechanisms informing the presynaptic terminal of the loss of postsynaptic receptivity remain poorly understood. Previous research at the mouse NMJ suggests that extracellular protons may function as a retrograde signal that triggers an upregulation of neurotransmitter output (measured by quantal content, QC) through the activation of acid-sensing ion channels (ASICs). We further investigated the pH-dependency of PHP in an ex-vivo mouse muscle preparation. We observed that increasing the buffering capacity of the perfusion saline with HEPES abolishes PHP and that acidifying the saline from pH 7.4 to pH 7.2-7.1 increases QC, demonstrating the necessity and sufficiency of extracellular acidification for PHP. We then sought to uncover how the blockade of nAChRs leads to the pH decrease. Plasma-membrane calcium ATPase (PMCA), a calcium-proton antiporter, is known to alkalize the synaptic cleft following neurotransmission in a calcium-dependent manner. We hypothesize that since nAChR blockade reduces postsynaptic calcium entry, it also reduces the alkalizing activity of the PMCA, thereby causing acidosis, ASIC activation, and QC upregulation. In line with this hypothesis, we found that pharmacological inhibition of the PMCA with carboxyeosin induces QC upregulation and that this effect requires functional ASICs. We also demonstrated that muscles pre-treated with carboxyeosin fail to generate PHP. These findings suggest that reduced PMCA activity causes presynaptic homeostatic potentiation by activating ASICs at the mouse NMJ.

A fast passive Ca2+ efflux mediated by the (Ca2+ + Mg2+)-ATPase in reconstituted vesicles.

The (Ca2+ + Mg2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into phospholipid bilayers. The permeability of lipid bilayers to Co2+ and glucose was increased slightly by incorporation of the ATPase, and the permeability of mixed bilayers of phosphatidylethanolamine and phosphatidylcholine increased with increasing content of phosphatidylethanolamine both in the presence and absence of the ATPase. The presence of the ATPase, however, resulted in a marked increase in permeability to Ca2+, the permeability decreasing with increasing phosphatidylethanolamine content. Permeability to Ca2+ was found to be dependent on pH and the external concentrations of Mg2+ and Ca2+, was stimulated by adenine nucleotides but was unaffected by inositol trisphosphate. A kinetic model is presented for Ca2+ efflux mediated by the ATPase. It is shown that the kinetic parameters that describe Ca2+ efflux from vesicles of sarcoplasmic reticulum also describe efflux from the vesicles reconstituted from the purified ATPase and phosphatidylcholine. It is shown that the effects of phosphatidylethanolamine on efflux can be simulated in terms of changes in the rates of the transitions linking conformations of the ATPase with inward- and outward-facing Ca2+-binding sites, and that effects of phosphatidylethanolamine on the ATPase activity of the ATPase can also be simulated in terms of effects on the corresponding conformational transitions. We conclude that the ATPase can act as a specific pathway for Ca2+ efflux from sarcoplasmic reticulum.

Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic reticulum Ca(2+)-ATPase in a rat model of heart failure.

BACKGROUND: In heart failure, sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling and contractile dysfunction. We have previously shown that increasing SERCA2a expression by gene transfer improves ventricular function in a rat model of heart failure created by ascending aortic constriction. METHODS AND RESULTS: In this study, we tested the effects of gene transfer of SERCA2a on survival, left ventricular (LV) volumes, and metabolism. By 26 to 27 weeks after aortic banding, all animals developed heart failure (as documented by >25% decrease in fractional shortening) and were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a) or control virus (Ad.betagal-GFP) by use of a catheter-based technique. Sham-operated rats, uninfected or infected with either Ad.betagal-GFP or Ad.SERCA2a, served as controls. Four weeks after gene transfer, survival in rats with heart failure treated with Ad.betagal-GFP was 9%, compared with 63% in rats receiving Ad.SERCA2a. LV volumes were significantly increased in heart failure (0.64+/-0.05 versus 0.35+/-0.03 mL, P<0.02). Overexpression of SERCA2a normalized LV volumes (0.46+/-0.07 mL) in the failing hearts. (31)P NMR analysis showed a reduced ratio of phosphocreatine to ATP content in failing+Ad.betagal-GFP compared with sham+Ad.betagal-GFP (0.82+/-0.13 versus 1.38+/-0.14, P<0.01). Overexpression of SERCA2a in failing hearts improved the phosphocreatine/ATP ratio (1.23+/-0.28). CONCLUSIONS: In this study, we show that unlike inotropic agents that improve contractile function at the expense of increased mortality and worsening metabolism, gene transfer of SERCA2a improves survival and the energy potential in failing hearts.

The responses to manipulation of extracellular and intracellular calcium are altered in the streptozotocin-diabetic rat colon and ileum.

Studies were performed to see if alterations in Ca2+ homeostasis underlie the gastrointestinal motility complications seen in many diabetic patients. Experiments were performed on colonic and ileal tissues taken from streptozotocin-induced diabetic and control rats. Diabetes caused alterations in the responses of the tissues to Ca2+ manipulation but these differed between the colon and ileum. In the colon a small but not significant increase in contractile responses to CaCl2 was observed in diabetic tissues, whereas the responses of the ileum were depressed relative to those of the controls. In contrast, responses of the diabetic ileum to the Ca2+ channel agonist Bay K8644 were greater than those of the controls, whilst the agonist failed to contract the colon. Similarly, the Ca2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid, produced contractions which were greater in diabetic ileal tissues. Thus, alterations in the responses of the diabetic gut to Ca2+ manipulation are complex, and also tissue-specific.

Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors.

Estrogen deficiency is associated with bone loss, and estrogen replacement is an effective treatment of this osteoporotic process. This study examines the early (5-120 s) effects of 17 beta-estradiol on the intracellular calcium and phospholipid metabolism in confluent female rat osteoblasts. The cytosolic free Ca2+ concentration ([Ca2+]i) was determined using fura-2/AM as Ca2+ probe. Cells were labeled with myo-[2-3H]inositol or [14C]arachidonic acid for inositol or lipid determination. Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) production were determined by either mass measurement or anion-exchange chromatography or by thin-layer chromatography, respectively. 17 beta-Estradiol (1 pM to 1 nM) increased [Ca2+]i in a biphasic manner within 10 s via Ca2+ influx from the extracellular milieu, as shown by the effects of the calcium chelator EGTA and the Ca2+ channel blockers nifedipine and verapamil, and via Ca2+ mobilization from the endoplasmic reticulum (ER), as shown by the effects of thapsigargin. 17 beta-Estradiol (1 pM to 1 nM) induced a biphasic and concomitant increase in IP3 and DAG formation. Estradiol immobilized on bovine serum albumin (BSA) [E-(O-carboxymethyl)oxime BSA] and its derivative (O-carboxymethyl)oxime rapidly increased ([Ca2+]i, IP3, and DAG and were full agonists, although they were less potent than the free estradiol. They had the same action time course and acted via Ca2+ influx and Ca2+ mobilization from ER. Tamoxifen, a potent inhibitor of genomic steroid responses, did not block the rapid increase in Ca2+, IP3, and DAG induced by estradiol. Finally, inhibitor of phospholipase C (neomycin) and pertussis toxin abolished the effects of 17 beta-estradiol on IP3 and DAG formation. These results suggest that female rat osteoblasts bear non-genomic unconventional cell surface receptors for estradiol, belonging to the class of the membrane receptors coupled to a phospholipase C via a pertussis toxin-sensitive G protein.

Ca(2+) oscillations regulated by Na(+)-Ca(2+) exchanger and plasma membrane Ca(2+) pump induce fluctuations of membrane currents and potentials in human mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (hMSCs) have the potential to differentiate into several types of cells. We have demonstrated spontaneous [Ca(2+)](i) oscillations in hMSCs without agonist stimulation, which result primarily from release of Ca(2+) from intracellular stores via InsP(3) receptors. In this study, we further investigated functions and contributions of Ca(2+) transporters on plasma membrane to generate [Ca(2+)](i) oscillations. In confocal Ca(2+) imaging experiments, spontaneous [Ca(2+)](i) oscillations were observed in 193 of 280 hMSCs. The oscillations did not sustain in the Ca(2+) free solution and were completely blocked by the application of 0.1mM La(3+). When plasma membrane Ca(2+) pumps (PMCAs) were blocked with blockers, carboxyeosin or caloxin, [Ca(2+)](i) oscillations were inhibited. Application of Ni(2+) or KBR7943 to block Na(+)-Ca(2+) exchanger (NCX) also inhibited [Ca(2+)](i) oscillations. Using RT-PCR, mRNAs were detected for PMCA type IV and NCX, but not PMCA type II. In the patch clamp experiments, Ca(2+) activated outward K(+) currents (I(KCa)) with a conductance of 170+/-21.6pS could be recorded. The amplitudes of I(KCa) and membrane potential (V(m)) periodically fluctuated liked to [Ca(2+)](i) oscillations. These results suggest that in undifferentiated hMSCs both Ca(2+) entry through plasma membrane and Ca(2+) extrusion via PMCAs and NCXs play important roles for [Ca(2+)](i) oscillations, which modulate the activities of I(KCa) to produce the fluctuation of V(m).

Gene silencing of selected calcium-signalling molecules in a Drosophila cell line using double-stranded RNA interference.

Using the Drosophila melanogaster S2 cell line, stably expressing a cloned muscarinic acetylcholine receptor (AChR), DM1, we have applied gene silencing by double-stranded RNA interference (RNAi) to knock down gene products involved in DM1-mediated calcium signalling. We have shown that RNAi knock down of either the inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P(3)R), or the SERCA calcium pump in the S2-DM1 cells blocks the increase in intracellular calcium concentration ([Ca(2+)](i)) resulting from activation of the DM1 receptor by 100 microM carbamylcholine (CCh). When RNAi designed to knock down the ryanodine receptor (RyR) was tested, there was no change in the calcium increase detected in response to CCh, consistent with a failure to detect RyRs in S2-DM1 cells using RT-PCR. A combination of RNAi and calcium imaging has provided a direct demonstration of key roles for the Ins(1,4,5)P(3)R and the SERCA pump in the response to DM1 receptor activation.Thus, we show that silencing of individual genes by RNAi in a well characterised Drosophila S2 cell line offers experimental opportunities for cell-signalling studies. Future investigations with RNAi libraries taking full advantage of the wealth of new information available from sequencing the Drosophila genome, may help identify novel components of cell-signalling pathways and functionally linked gene products.

Ca2+-induced Ca2+ release by activation of inositol 1,4,5-trisphosphate receptors in primary pancreatic beta-cells.

The effect of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition on the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) was studied in primary insulin-releasing pancreatic beta-cells isolated from mice, rats and human subjects as well as in clonal rat insulinoma INS-1 cells. In Ca(2+)-deficient medium the individual primary beta-cells reacted to the SERCA inhibitor cyclopiazonic acid (CPA) with a slow rise of [Ca(2+)](i) followed by an explosive transient elevation. The [Ca(2+)](i) transients were preferentially observed at low intracellular concentrations of the Ca(2+) indicator fura-2 and were unaffected by pre-treatment with 100 microM ryanodine. Whereas 20mM caffeine had no effect on basal [Ca(2+)](i) or the slow rise in response to CPA, it completely prevented the CPA-induced [Ca(2+)](i) transients as well as inositol 1,4,5-trisphosphate-mediated [Ca(2+)](i) transients in response to carbachol. In striking contrast to the primary beta-cells, caffeine readily mobilized intracellular Ca(2+) in INS-1 cells under identical conditions, and such mobilization was prevented by ryanodine pre-treatment. The results indicate that leakage of Ca(2+) from the endoplasmic reticulum after SERCA inhibition is feedback-accelerated by Ca(2+)-induced Ca(2+) release (CICR). In primary pancreatic beta-cells this CICR is due to activation of inositol 1,4,5-trisphosphate receptors. CICR by ryanodine receptor activation may be restricted to clonal beta-cells.

Effect of the Ca(2+)-ATPase inhibitor, cyclopiazonic acid, on electromechanical coupling in the guinea-pig ureter.

1. We have investigated the effect of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor, cyclopiazonic acid (CPA), on electromechanical coupling in the guinea-pig ureter. All experiments were performed in capsaicin-pretreated (10 microM for 15 min) ureters to prevent the release of sensory neuropeptides from afferent nerves. 2. In organ bath experiments, electrical field stimulation (EFS, 10 Hz for 1 s, 5 ms pulse width, 60 V) produced tetrodotoxin- (1 microM) resistant phasic contractions which were enhanced by Bay K 8644 (1 microM) and abolished by nifedipine (10-30 microM). 3. CPA (10 microM) enhanced the EFS-evoked contractions both in the absence and presence of Bay K 8644. The effect of CPA was concentration-dependent between 1 and 30 microM. The response to 10 microM CPA was biphasic: the maximal enhancement (58 +/- 3% increase) was observed within 10-20 min from CPA administration, followed by a decline to a new steady state (25 +/- 5% increase over baseline) at 50-60 min. The effect of CPA was reversed by washout. 4. Ryanodine (100 microM) produced a prompt enhancement of the EFS-evoked contractions of the guinea-pig ureter, which peaked at 42 +/- 3% increase over baseline; the co-administration of CPA (10 microM) and ryanodine (100 microM) produced a peak effect (60 +/- 8% enhancement) which was not different from that produced by CPA alone. With either ryanodine alone or ryanodine plus CPA, the enhancement of the EFS-induced contractions was biphasic, showing a time-course similar to that observed with CPA alone. Tetraethylammonium (10 mM) produced a significantly larger effect (93 +/- 13% increase over baseline) and its effect was sustained throughout the 60 min observation period. 5. In the presence of Bay K 8644, superfusion for 30 min with a low Na+ medium (60% of extracellular Na+ replaced by Li+ or choline) reduced the amplitude of EFS-evoked contractions by 20-35%. In both Li(+)- and choline-substituted media, spontaneous activity developed during superfusion with low Na+ Krebs solution which was suppressed by 10 microM nifedipine. CPA (10 microM) produced a marked enhancement of the EFS-evoked contractions in low-Na+ medium (both Li(+)- and choline-substituted) and this effect was sustained throughout the 60 min observation period. 6. In the absence of Bay K 8644, the response of the ureter smooth muscle to EFS is characterized by a refractory period: an interval of about 30 s was required between two applied stimuli to produce a second response comparable in size to that elicited by the first stimulus. CPA (10 micro M, 10-20 min before)markedly reduced the refractory period of the guinea-pig ureter to EFS.7. CPA (10 micro M, 30-60 min before) increased the phasic component of contraction produced by 80 m MKCl. The tonic component of the response to KCl was slightly but not significantly reduced by CPA,and a 'hump' in the tonic contraction was observed at 1-2 min from addition of KCl.8. In sucrose gap experiments, 10 micro M CPA produced a sustained depolarization of the membrane and reduced the latency between application of electrical stimuli and onset of the action potential; these effects were maintained throughout the 60 min superfusion with CPA. CPA also transiently prolonged the plateau phase of the action potential and increased the peak amplitude of contraction: these effects peaked at about 10-20 min from start of superfusion with CPA and then declined. At the peak of its enhancing effect on contraction amplitude, CPA prolonged the contractile phase of the contraction relaxation cycle.9.Superfusion with a low-Na, choline-substituted Krebs solution produced a reversible membrane depolarization. In the presence of Bay K 8644 (1 micro M), action potentials and phasic contractions were superimposed on this depolarization which were abolished by nifedipine (1O micro M).10. These findings indicate that CPA augments the excitability and affects the contraction-relaxation cycle of the smooth muscle of the guinea-pig ureter, implying a role for sarcoplasmic reticulum Ca2+-ATPase in the regulation of electromechanical coupling. The effects of CPA resemble those produced by ryanodine and the effect of the two agents on the amplitude of contractions is non-additive.It appears that following blockade of the CPA-sensitive SR Ca2+ pump, other mechanism(s) may come into action to reduce intracellular Ca2+. The Na+/Ca2+ exchanger could be involved in the compensatory changes responsible for the fading of the response to CPA.

Effects of thapsigargin, an intracellular calcium-mobilizing agent, on synthesis and secretion of cartilage collagen and proteoglycan.

The calcium-mobilizing agents thapsigargin and 2,5-di-(tert-butyl)-1,4- benzohydroquinone were shown to markedly elevate the intracellular calcium concentration of chick embryo chondrocytes in a dose-dependent manner. Under these conditions, the metabolism of macromolecules was variably affected. The synthesis and secretion of protein in general, and of collagen in particular, were significantly inhibited; in contrast, proteoglycan synthesis (but not glycosaminoglycan synthesis) was inhibited, whereas secretion was unaffected. Flunarizine, which prevented the thapsigargin-induced intracellular calcium elevation, and EGTA, which caused only a transient thapsigargin-induced intracellular calcium elevation, did not reverse these alterations. It was concluded, therefore, that the observed effects of thapsigargin and 2,5-di-(tert-butyl)-1,4-benzohydroquinone on chondrocyte macromolecule metabolism were not related to the ability of these drugs to increase the cytosolic free calcium concentration but may have been due to the specific depletion of the calcium sequestered in the endoplasmic reticulum. The differential effect of these drugs on protein and proteoglycan secretion suggests that the intracellular trafficking of these two classes of macromolecules may be controlled independently.

Induction of hepatic metallothionein synthesis by endoplasmic reticulum stress in mice.

Metallothionein (MT) is a small sulfhydryl-rich protein whose levels are elevated by various inducers of organelle stresses, such as nuclear stress (cisplatin), mitochondrial stress (antimycin A, 2,4-dinitrophenol) and lysosomal stress (paraquat). Although abnormal folding of protein in the endoplasmic reticulum (ER) causes ER stress, induction of MT synthesis by ER stress has never been investigated. In this study, we examined the induction of MT by an inducer of ER stress, tunicamycin (Tun), which induces ER stress by inhibiting N-linked glycosylation of protein in the ER. Administration of Tun (0.5-1.5 mg/kg, sc) increased hepatic MT levels in C57BL/6J mice (3.1-fold). The maximal increase in hepatic MT was observed 48-96 h after the administration of Tun (1.0 mg/kg). Expressions of MT-I, II and glucose-regulated protein 78 (Bip/GRP78), which is a molecular chaperone induced by ER stress, mRNA were also detected by administration of Tun. Thapsigargin (Thap), a generator of ER stress by inhibiting ER Ca(2+)-ATPase, also increased both hepatic MT levels and expression of MT-I and -II mRNA. The level of expression of Bip/GRP78 mRNA induced by Tun administration in MT-null mice was greater than that in wild-type mice. Taken together, these findings suggest that inhibitors of ER are potent inducers of MT.

Simulation of Ca2+ release from the sarcoplasmic reticulum with three-dimensional sarcomere model in cardiac muscle.

A simulation of some basic features of Ca(2+) release from the sarcoplasmic reticulum (SR) in cardiac muscle was made with a model based on the mechanism of Ca(2+)-induced Ca(2+)-release. The half-sarcomere modeled as a circular cylinder was divided into 20 annular elements in the radial, 50 slices in the axial, and 125 slices in the azimuthal direction. The cylindrical surface of the sarcomere was covered by a layer of the SR. The rate of Ca(2+) release from the terminal sac (TS) is proportional to the product of the open probability of the Ca(2+) release channel and the difference of [Ca(2+)] between the TS and an element facing the TS. Ca(2+) moves from element to element by simple diffusion and is taken up by the tubular SR via Ca(2+)-ATPase. Ca(2+) influx (I(ca)) to trigger the TS Ca(2+) release was introduced to either a single element facing the TS (local I(ca)) or to 20 elements aligned at the level of the Z-line (uniform I(ca)). The simulation showed that with both types of I(ca), TS Ca(2+) release is smoothly graded over a wide range of I(ca) with the TS moderately loaded with Ca(2+). The gain determined by dividing the total amount of TS Ca(2+) release by I(ca) was greater with local than with uniform I(ca). Mechanical alternans was simulated with both the local and uniform I(ca) with an appropriate rate of Ca(2+) replenishment to the TS. A Ca(2+) wave was simulated with a model consisting of 8 longitudinally consecutive sarcomeres with TS heavily loaded with Ca(2+). Thus the present model accounted for graded TS Ca(2+) release, mechanical alternans, and Ca(2+) wave in cardiac muscle at the same time.

Hyperthyroidism causes mechanical insufficiency of myocardium with possibly increased SR Ca2+-ATPase activity.

Hyperthyroidism is known to affect multiple organ functions, and thyroid hormone has been known to improve myocardial function in a failing heart. The purpose of this study is to elucidate the functional and metabolic effects of thyroid hormone on myocardium in a rat model exposed to long-term excess thyroid hormone, particularly focusing on the SR Ca(2+)-ATPase (SERCA2) function. 3,5,3'-Triiodo-L-thyronine (T3), or the vehicle, was subcutaneously given for 4 weeks (T3 and control [C] group). Bolus I.V. Thapsigargin (TG) was used to test the SERCA2 function (C-TG and T3-TG) in Langendorff perfused heart. Myocardial functions such as LV-developed pressure (LVDP; mmHg), +/- dP/dt (mmHg/s), tau (ms), and oxygen consumption (MVO(2); ml/min/g wt) were measured. SERCA2 and GLUT4 protein level were also evaluated by Western immunoblotting. Left ventricle to body weight (LV/BW) ratio was significantly higher in the T3 group. Both negative dP/dt and tau were significantly decreased by TG. It is interesting that the decrement of negative dP/dt and tau attained by TG was significantly larger in the hyperthyroid group (T3-TG) than in a normal heart (C-TG). SERCA2 and GLUT4 protein levels were not significantly different between control and the T3 group. We conclude that prolonged exposure to thyroid hormone causes hypertrophy of the myocardium and an augmentation of the SR Ca(2+) ATPase activity. Care must be taken in hyperthyroid heart during the ischemia-reperfusion process where the SRECA2 function is inhibited.

Effect of low salinity on cadmium accumulation and calcium homeostasis in the shore crab (Carcinus maenas) at fixed free Cd2+ concentrations.

Increased Cd toxicity at low salinity has been attributed to increased free Cd2+ ion concentration ([Cd2+]sw), but transfer to dilute seawater also stimulates physiological ionic regulation in crabs. In this study, Cd accumulation and Ca homeostasis in the shore crab (Carcinus maenas) were explored at fixed [Cd2+]sw to reveal the physiological events during sublethal Cd exposure. Crabs were exposed to 3.4 or 34 microg/L [Cd2+], in both 100% seawater (SW) and 33% SW for up to 10 d and sampled for hemolymph composition as well as gill and hepatopancreas Ca, Cd, and Ca-ATPase activity. Cadmium exposure ameliorated the expected fall in hemolymph osmotic pressure and NaCl at low salinity and generally protected tissue Ca from decline. Cadmium exposure alone (within salinity) inhibited Ca-ATPase, but this was offset by stimulation of Ca-ATPase at low salinity. The Ca-ATPase activity in the anterior and posterior gills showed different responses to Cd/low salinity stress. Crabs were more sensitive to a 10-fold increase in [Cd2+]sw at low salinity. Overall, we conclude that exposure to a fixed sublethal [Cd2+]sw reveals a compensatory physiological response that is driven primarily by salinity rather than Cd2+ free ion concentration. Physiological responses are therefore important during low-level Cd exposure in dilute seawater.

Voltage-gated channels and calcium homeostasis in mammalian rod photoreceptors.

Recent reports on rod photoreceptor neuroprotection by Ca2+ channel blockers have pointed out the need to assess the effect of these blockers on mammalian rods. However, in mammals, rod electrophysiological characterization has been hampered by the small size of these photoreceptors, which were instead extensively studied in nonmammalian vertebrates. To further characterize ionic conductances and to assess the pharmacology of Ca2+ channels in mammalian rods, freshly dissociated pig rod photoreceptors were recorded with the whole cell patch-clamp technique. Rod cells expressed 1) a hyperpolarization-activated inward-rectifying conductance (I(h)) sensitive to external Cs+; 2) a sustained outward K+ current (I(K)) sensitive to tetraethylammonium; 3) a sustained voltage-gated Ca2+ current (I(Ca)) sensitive to benzothiazepine (diltiazem) and phenylalkylamine (verapamil) derivatives; 4) a Ca(2+)-activated Cl- current (I(Cl(Ca))); and 5) a plasma membrane Ca(2+)-ATPase. The Ca2+ current showed a range of activation from positive potentials to -60 mV with a maximum between -30 and -20 mV. In contrast to other L-type Ca2+ channels, rod Ca2+ channels were blocked at similar and relatively high concentrations by the diltiazem isomers and verapamil. The biphasic dose-response for D-diltiazem confirmed the low sensitivity of Ca2+ channels for the molecule. The ATPase, which was localized at the axon terminal, was found to contribute to Ca2+ extrusion. These results suggest that the electrophysiological features of rod photoreceptors had been preserved during evolution from nonmammalian vertebrates to mammals. This work indicates further that mammalian rods express nonclassic L-type Ca2+ channels, showing a low sensitivity to the diltiazem isomers used in neuroprotective studies.

Is ouabain produced by the adrenal gland?

1. Ouabain or a related stereoisomer, termed endogenous ouabain, has been identified in adrenal cortex tissue and culture medium from adrenocortical cells. 2. Angiotensin II and adrenocorticotropin, the main activators of aldosterone secretion from adrenal glomerulosa cells appear to increase the production of this compound. 3. The purpose of this review is to briefly discuss recent available experimental evidence suggesting that endogenous ouabain is secreted by the zona glomerulosa of the adrenal gland.

Thyroid hormone-induced alterations in phospholamban-deficient mouse hearts.

Alterations in the expression levels of the sarcoplasmic reticulum (SR) Ca2+-ATPase and its regulator, phospholamban, have been implicated in the effects of thyroxine hormone on cardiac function. To determine the role of phospholamban in these effects, hypothyroidism and hyperthyroidism were induced in phospholamban-deficient mice and their isogenic wild types. Hypothyroidism resulted in significant decreases of left ventricular contractility, which could be moderately stimulated by increases in preload or afterload, in both phospholamban-deficient and wild-type mice. However, the basal contractile parameters in hypothyroid phospholamban-deficient hearts were at least as high as those exhibited by hyperthyroid wild-type hearts. In hyperthyroidism, there was no further enhancement of the hyperdynamic contractile parameters in phospholamban-deficient hearts, although the wild-type hearts exhibited significantly increased contractile function compared with their respective euthyroid groups. Furthermore, increases in preload or afterload did not enhance contractility in either phospholamban-deficient or wild-type hyperthyroid hearts. Examination of the relative tissue levels of cardiac SR Ca2+-ATPase revealed increases in hyperthyroidism and decreases in hypothyroidism compared with euthyroidism, and these changes were similar between phospholamban-deficient and wild-type hearts. An opposite trend was observed for phospholamban expression levels in the wild-type group, which were depressed in hyperthyroid hearts but increased in hypothyroid hearts. These findings indicate that (1) thyroid hormones induce similar changes in the cardiac SR Ca2+-ATPase levels in either the presence or absence of phospholamban, (2) the thyroxine-induced increases in SR Ca2+-ATPase levels are not associated with any further stimulation of the hyperdynamic cardiac function in phospholamban-deficient mice, and (3) the decreased contractile parameters in hypothyroid phospholamban-deficient hearts associated with decreases in SR Ca2+-ATPase levels and myosin heavy chain isoform switches are at least as high as those of the stimulated hyperthyroid wild-type hearts. Thus, alterations in the phospholamban level or its activity may be a critical determinant of the contractile responses to altered thyroid states in the mammalian heart.

Receptors and second messengers involved in long-term depression in rat cerebellar slices in vitro: a reappraisal.

In patch-clamped Purkinje cells (PCs), bath application of the ionotropic glutamate receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) prevents induction of long-term depression (LTD) of parallel fibre (PF)-mediated EPSPs by a pairing protocol between Ca2+ spike firing and PF stimulation whereas bath application of (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG), a metabotropic glutamate (mGLU) receptor antagonist, does not. On the other hand, LTD can be also induced by pairing direct depolarization of PCs with activation of mGLU receptors by 1S,3R-aminocyclopentyl-dicarboxylate (1S,3R-ACPD), even in the presence of CNQX. In this case, LTD induction is not consistently blocked by bath application of the nitric oxide synthase inhibitor, NG-methyl-L-arginine (L-NMMA), whereas it is strongly blocked when the protein kinase C inhibitor peptide 19-36 is dialysed into PCs. These results are at variance with LTD induced by a pairing protocol between Ca2+ spikes and PF-mediated EPSPs which depends to the same extent on both cascades. Finally, thapsigargin, which depletes most intracellular Ca2+ pools, does not block induction of LTD by a pairing protocol between Ca2+ spikes and PF-mediated EPSPs whereas it prevents the induction of LTD depending on strong mGLU receptor activation.