Palmitoyl carnitine increases intracellular calcium in adult rat cardiomyocytes.

Earlier studies have demonstrated that palmitoyl carnitine (PC), a long chain acyl carnitine, accumulates in the ischemic myocardium. Although perfusion of hearts with PC is known to induce contractile dysfunction which resembles ischemic contracture, the mechanisms underlying this derangement are not clear. In this study, we examined the effect of exogenous PC on the intracellular concentration of calcium ([Ca(2+)](i)) in freshly isolated cardiomyocytes from adult rat hearts. The results showed that PC elevated [Ca(2+)](i)in a dose-dependent (5-20 microm) manner; 15 microm PC evoked a marked and reversible increase in [Ca(2+)](i)without having any significant action on cell viability. The PC (15 microm)-induced increase in [Ca(2+)](i)was slightly depressed but delayed in the absence of extracellular Ca(2+). Pre-incubation of cardiomyocytes with sarcolemmal (SL) l -type Ca(2+)-channel blockers, verapamil or diltiazem, and inhibitors of SL Na(+)-Ca(2+)exchanger such as Ni(2+)or amiloride, depressed the PC-evoked increase in [Ca(2+)](i)significantly. Ouabain, a Na(+)-K(+)ATPase inhibitor, and low concentrations of extracellular Na(+)enhanced the PC-induced increase in [Ca(2+)](i). Depletion of the sarcoplasmic reticulum (SR) Ca(2+)stores by low micromolar concentrations of ryanodine (a SR Ca(2+)-release channel activator) or by thapsigargin (a SR Ca(2+)-pump ATPase inhibitor) depressed the PC-mediated increase in [Ca(2+)](i). Combined blockade of the l -type Ca(2+)channel, Na(+)-Ca(2+)exchanger and the SR Ca(2+)-pump had an additive inhibitory effect on the PC response. These observations suggest that the PC-induced increase in [Ca(2+)](i)is dependent on both Ca(2+)-influx from the extracellular space and Ca(2+)-release from the SR stores. Thus, the accumulation of PC in the myocardium may be partly responsible for the occurrence of intracellular Ca(2+)overload in ischemic heart.     

Direct evidence for calcium conductance of hyperpolarization-activated cyclic nucleotide-gated channels and human native If at physiological calcium concentrations

AIMS: The hyperpolarization-activated cyclic nucleotide-gated (HCN) current I(f)/I(HCN) is generally thought to be carried by Na(+) and K(+) under physiological conditions. Recently, Ca(2+) influx through HCN channels has indirectly been postulated. However, direct functional evidence of Ca(2+) permeation through I(f)/I(HCN) is still lacking. METHODS AND RESULTS: To possibly provide direct evidence of Ca(2+) influx through I(HCN)/I(f), we performed inside-out and cell-attached single-channel recordings of heterologously expressed HCN channels and native rat and human I(f), since Ca(2+)-mediated I(f)/I(HCN) currents may not readily be recorded using the whole-cell technique. Original current traces demonstrated HCN2 Ca(2+) inward currents upon hyperpolarization with a single-channel amplitude of -0.87+/-0.06 pA, a low open probability of 3.02+/-0.48% (at -110 mV, n=6, Ca(2+) 2 mmol/L), and a Ca(2+) conductance of 8.9+/-1.2 pS. I(HCN2-Ca2+) was significantly activated by the addition of cAMP with an increase in the open probability and suppressed by the specific I(f) inhibitor ivabradine, clearly confirming that Ca(2+) influx indeed was conducted by HCN2 channels. Changing [Na(+)] (10 vs. 100 mmol/L) in the presence or absence of 2 mmol/L Ca(2+) caused a simple shift of the reversal potential along the voltage axis without significantly affecting Na(+)/Ca(2+) conductance, whereas the K(+) conductance of HCN2 increased significantly in the absence of external Ca(2+) with increasing K(+) concentrations. The mixed K(+)-Ca(2+) conductance, however, was unaffected by the external K(+) concentration. Notably, we could also record hyperpolarization-activated Ca(2+) permeation of single native I(f) channels in neonatal rat ventriculocytes and human atrial myocytes in the presence of blockers for all known cardiac calcium conduction pores (Ca(2+) conductance of human I(f), 9.19+/-0.34 pS; amplitude, -0.81+/-0.01 pA; open probability, 1.05+/-0.61% at -90 mV). CONCLUSION: We directly show Ca(2+) permeability of native rat and, more importantly, human I(f) at physiological extracellular Ca(2+) concentrations at the physiological resting membrane potential. This might have particular implications in diseased states with increased I(f) density and HCN expression.     

Reduction of Ca(2+) channel activity by hypoxia in human and porcine coronary myocytes.

OBJECTIVE: Oxygen (O(2)) tension is a major regulator of blood flow in the coronary circulation. Hypoxia can produce vasodilation through activation of ATP regulated K(+) (K(ATP)) channels in the myocyte membrane, which leads to hyperpolarization and closure of voltage-gated Ca(2+) channels. However, there are other O(2)-sensitive mechanisms intrinsic to the vascular smooth muscle since hypoxia can relax vessels precontracted with high extracellular K(+), a condition that prevents hyperpolarization following opening of K(+) channels. The objective of the present study was to determine whether inhibition of Ca(2+) influx through voltage-dependent channels participates in the response of coronary myocytes to hypoxia. METHODS: Experiments were performed on porcine anterior descendent coronary arterial rings and on enzymatically dispersed human and porcine myocytes of the same artery. Cytosolic [Ca(2+)] was measured by microfluorimetry and whole-cell currents were recorded with the patch clamp technique. RESULTS: Hypoxia (O(2) tension approximately 20 mmHg) dilated endothelium-denuded porcine coronary arterial rings precontracted with high K(+) in the presence of glibenclamide (5 microM), a blocker of K(ATP) channels. In dispersed human and porcine myocytes, low O(2) tension decreased basal cytosolic [Ca(2+)] and transmembrane Ca(2+) influx independently of K(+) channel activation. In patch clamped cells, hypoxia reversibly inhibited L-type Ca(2+) channels. RT-PCR indicated that rHT is the predominant mRNA variant of the alpha(1C) Ca(2+) channel subunit in human coronary myocytes. CONCLUSION: Our study demonstrates, for the first time in a human preparation, that voltage-gated Ca(2+)channels in coronary myocytes are under control of O(2) tension.     

Hyperpolarization-activated calcium channels at the tip of Arabidopsis root hairs

The root hair elongative growth phase ("tip growth"), like that of other tip-growing systems such as pollen tubes, algal rhizoids, and fungal hyphae, is associated with an apex-high cytosolic free calcium ([Ca(2+)](c)) gradient generated by a local Ca(2+) influx at the tip. This gradient has been shown to be a fundamental regulator of tip growth. Here, we have performed patch-clamp experiments at root hair apices of Arabidopsis thaliana (after localized cell wall laser ablation) to characterize the plasma membrane Ca(2+) channels implicated in the tip Ca(2+) influx. We have identified a hyperpolarization-activated Ca(2+) conductance. This conductance is selective for Ca(2+) over K(+) and Cl(-) (P(Ca)/P(K) = 15; P(Ca)/P(Cl) = 25) and is fully blocked by < 100-microM trivalent cations (La(3+), Al(3+), Gd(3+)). The selectivity sequence among divalent cations (determined by comparisons of the channel unitary conductance) is Ba(2+) > Ca(2+) (22 pS in 10 mM) approximately Mg(2+) > Mn(2+). This conductance was operative at typical growing hair apical resting membrane potentials. Moreover, it was seen to be down-regulated in growing hair subapical regions, as well as at the tip of mature hairs (known not to exhibit Ca(2+) influx). We therefore propose that this inward-rectifying Ca(2+) conductance is inherently involved in the apical Ca(2+) influx of growing hairs. The observed enhancement of the conductance by increased [Ca(2+)](c) may form part of a positive feedback system for continued apical Ca(2+) influx during tip growth.     

Epoxyeicosatrienoic acids increase intracellular calcium concentration in vascular smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid. They are potent endogenous vasodilator compounds produced by vascular cells, and EET-induced vasodilation has been attributed to activation of vascular smooth muscle cell (SMC) K(+) channels. However, in some cells, EETs activate Ca(2+) channels, resulting in Ca(2+) influx and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). We investigated whether EETs also can activate Ca(2+) channels in vascular SMC and whether the resultant Ca(2+) influx can influence vascular tone. The 4 EET regioisomers (1 micromol/L) increased porcine aortic SMC [Ca(2+)](i) by 52% to 81%, whereas arachidonic acid, dihydroxyeicosatrienoic acids, and 15-hydroxyeicosatetraenoic acid (1 micromol/L) produced little effect. The increases in [Ca(2+)](i) produced by 14,15-EET were abolished by removal of extracellular Ca(2+) and by pretreatment with verapamil (10 micromol/L), an inhibitor of voltage-dependent (L-type) Ca(2+) channels. 14,15-EET did not alter Ca(2+) signaling induced by norepinephrine and thapsigargin. When administered to porcine coronary artery rings precontracted with a thromboxane mimetic, 14,15-EET produced relaxation. However, when administered to rings precontracted with acetylcholine or KCl, 14,15-EET produced additional contractions. In rings exposed to 10 mmol/L KCl, a concentration that did not affect resting ring tension, 14,15-EET produced small contractions that were abolished by EGTA (3 mmol/L) or verapamil (10 micromol/L). These observations indicate that 14,15-EET enhances [Ca(2+)](i) influx in vascular SMC through voltage-dependent Ca(2+) channels. This 14,15-EET-induced increase in [Ca(i)(2+)] can produce vasoconstriction and therefore may act to modulate EET-induced vasorelaxation.     

Ghrelin reduces voltage-gated potassium currents in GH3 cells via cyclic GMP pathways

Ghrelin is an endogeneous growth hormone secretagogue (GHS) causing release of GH from pituitary somatotropes through the GHS receptor. Secretion of GH is linked directly to intracellular free Ca²⁺ concentration ([Ca²⁺]_i), which is determined by Ca²⁺ influx and release from intracellular Ca²⁺ storage sites. Ca²⁺ influx is via voltage-gated Ca²⁺ channels, which are activated by cell depolarization. Membrane potential is mainly determined by transmembrane K⁺ channels. The present study investigates the in vitro effect of ghrelin on membrane voltage-gated K⁺ channels in the GH₃ rat somatotrope cell line. Nystatin-perforated patch clamp recording was used to record K⁺ currents under voltage-clamp conditions. In the presence of Co²⁺ (1 mM, Ca²⁺ channel blocker) and tetrodotoxin (1 μM, Na⁺ channel blocker) in the bath solution, two types of voltage-gated K⁺ currents were characterized on the basis of their biophysical kinetics and pharmacological properties. We observed that transient K⁺ current (I _A) represented a significant proportion of total K⁺ currents in some cells, whereas delayed rectifier K⁺ current (I _K) existed in all cells. The application of ghrelin (10 nM) reversibly and significantly decreased the amplitude of both I _A and I _K currents to 48% and 64% of control, respectively. Application of apamin (1 μM, SK channel blocker) or charybdotoxin (1 μM, BK channel blocker) did not alter the K⁺ current or the response to ghrelin. The ghrelin-induced reduction in K⁺ currents was not affected by PKC and PKA inhibitors. KT5823, a specific PKG inhibitor, totally abolished the K⁺ current response to ghrelin. These results suggest that ghrelininduced reduction of voltage-gated K⁺ currents in GH₃ cells is mediated through a PKG-dependent pathway. A decrease in voltage-gated K⁺ currents may increase the frequency, duration, and amplitude of action potentials and contribute to GH secretion from somatotropes.     

Structure and function of multiple Ca2+-binding sites in a K+ channel regulator of K+ conductance (RCK) domain

Regulator of K(+) conductance (RCK) domains control the activity of a variety of K(+) transporters and channels, including the human large conductance Ca(2+)-activated K(+) channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca(2+)-gated K(+) channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca(2+). Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca(2+)-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca(2+)-binding sites, resulting in a stoichiometry of 24 Ca(2+) ions per channel, is consistent with the steep relation between [Ca(2+)] and MthK channel activity. Comparison of Ca(2+)-bound and unliganded RCK domains suggests a physical mechanism for Ca(2+)-dependent conformational changes that underlie gating in this class of channels.     

Sodium-calcium exchange in mammalian myocardium: the effects of lithium

Studies were carried out to investigate the effect of lithium (Li) on mechanical function and on sodium (Na) and calcium (Ca) exchange in the isolated, arterially perfused interventricular septum of the rabbit. Perfusion of the septum with solutions containing zero potassium (0 K) and either 142 m Na or 36 m Na replaced by sucrose caused large increments in Ca uptake and the development of contracture. If, however, Li was substituted for Na instead of sucrose, both Ca uptake and contracture were markedly reduced. Despite the fact that Li substitution caused progressive loss of contracture, and sucrose substitution an increase of contracture (under 0 K perfusion conditions) neither substitution altered the rate of ⁴⁵Ca efflux. ⁴⁷Ca uptake experiments showed that substitution of Li for Na (no previous sucrose substitution) in 0 K perfusion, thereby producing an increase in [Na]_i/[Na]₀ ratio, caused an increase in Ca uptake which reached a plateau within 30 min. However, when Li was substituted for sucrose (unchanged [Na]_i/[Na]₀) the progressive net uptake of Ca associated with sucrose stopped immediately and a progressive net loss was induced. Three conclusions are drawn from these results: (1) Li blocks the contractures produced during a 0 K perfusion by decreasing both Ca uptake and Na efflux. (2) An increase in [Na]_i/[Na]₀ ratio induces an initial contracture and increased Ca uptake. (3) The progression of contracture and Ca uptake is dependent upon continued operation of the Na---Ca exchange system. This does not occur in the presence of Li. The substitution of Li for Na separates two facets of the Na---Ca relationship: (1) the effect of [Na]₀ on superficial Ca-binding, (2) the effect of [Na]_i on the Na---Ca “carrier”.     

Mechanism of contraction of rat isolated tail arteries by hyposmotic solutions

Contraction induced by hyposmotic swelling was examined in rat tail arteries mounted on a myograph containing a modified Krebs physiological saline solution (PSS) containing 50 mM mannitol (300 mosm/l). Hyposmotic swelling was induced by removing mannitol. In arteries having basal tone or arteries precontracted with K(+) or the thromboxane mimetic U-46619, removal of mannitol caused a concentration dependent contraction of rat tail arteries. Concurrent measurement of tension and intracellular calcium [Ca(2+)](i )in arteries loaded with fura-2 showed that both tension and [Ca(2+)](i) increased on exposure to a hyposmotic solution. Removal of endothelium or inhibition of nitric oxide and cyclooxygenase together did not affect contractile responses. Removal of extracellular Ca(2+) abolished the contractile response to hyposmotic solution and NiCl(2), a nonspecific inhibitor of Ca(2+) influx pathways, blocked the rise in [Ca(2+)](i) and tension in response to a hyposmotic solution. Verapamil and nisoldipine, inhibitors of Ca(v)1.2 (L-type) calcium channels significantly reduced the contractile response to a hyposmotic solution. Addition of NiCl(2) to nisoldipine caused an additional inhibition of the response to a hyposmotic solution. Inhibition of calcium release from the sarcoplasmic reticulum by ryanodine or cyclopiazonic acid (CPA) did not cause any change in the tension response to a hyposmotic solution. CPA did not significantly inhibit the response to a hyposmotic solution in the presence of N(G)-methyl-L-arginine, oxyhaemoglobin and indomethacin. We conclude that contraction induced by a hyposmotic solution is largely due to Ca(v)1.2 calcium channels although other Ca(2+) influx pathways also contribute.     

Dopamine inhibits basal prolactin release in pituitary lactotrophs through pertussis toxin-sensitive and -insensitive signaling pathways

Dopamine D2 receptors signal through the pertussis toxin (PTX)-sensitive G(i/o) and PTX-insensitive G(z) proteins, as well as through a G protein-independent, beta-arrestin/glycogen synthase kinase-3-dependent pathway. Activation of these receptors in pituitary lactotrophs leads to inhibition of prolactin (PRL) release. It has been suggested that this inhibition occurs through the G(i/o)-alpha protein-mediated inhibition of cAMP production and/or G(i/o)-betagamma dimer-mediated activation of inward rectifier K(+) channels and inhibition of voltage-gated Ca(2+) channels. Here we show that the dopamine agonist-induced inhibition of spontaneous Ca(2+) influx and release of prestored PRL was preserved when cAMP levels were elevated by forskolin treatment. We further observed that dopamine agonists inhibited both spontaneous and depolarization-induced Ca(2+) influx in untreated but not in PTX-treated cells. This inhibition was also observed in cells with blocked inward rectifier K(+) channels, suggesting that the dopamine effect on voltage-gated Ca(2+) channel gating is sufficient to inhibit spontaneous Ca(2+) influx. However, agonist-induced inhibition of PRL release was only partially relieved in PTX-treated cells, indicating that dopamine receptors also inhibit exocytosis downstream of voltage-gated Ca(2+) influx. The PTX-insensitive step in agonist-induced inhibition of PRL release was not affected by the addition of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, and lithium, an inhibitor of glycogen synthase kinase-3, but was attenuated in the presence of phorbol 12-myristate 13-acetate, which inhibits G(z) signaling pathway in a protein kinase C-dependent manner. Thus, dopamine inhibits basal PRL release by blocking voltage-gated Ca(2+) influx through the PTX-sensitive signaling pathway and by desensitizing Ca(2+) secretion coupling through the PTX-insensitive and protein kinase C-sensitive signaling pathway.     

Extracellular ATP stimulates estradiol secretion in rat Sertoli cells in vitro: modulation by external sodium

In this study, we examined the effects of extracellular ATP (ATPe) on [Ca(2+)](i), [Na(+)](i), plasma membrane potential changes and estradiol secretion in rat Sertoli cells. ATPe caused a rapid rise of [Ca(2+)](i) with an initial spike followed by a long lasting plateau. The first rapid spike was dependent on the release of Ca(2+) from internal stores as it also occurred in Ca(2+)-free medium while the long lasting plateau phase was dependent on Ca(2+) influx from the external medium. ATPe stimulated a rapid plasma membrane depolarization that was dependent on an influx of Na(+) from the external medium as demonstrated by plasma membrane potential monitoring in Na(+)-free medium and by [Na(+)](i) measurement with the Na(+)-sensitive fluorescent dye SBFI. ATPe stimulated estradiol secretion in a dose dependent manner and was fully dependent on the presence of Na(+) in the external medium while the presence of Ca(2+) was not necessary. Among the different nucleotides tested, only ATP, ATP-5'-[gamma-thio]triphosphate, UTP, alpha,beta-methylene-ATP were effective in stimulating estradiol secretion. These results demonstrate that rat Sertoli cells possess P2-purinergic receptors belonging to the P2X and P2Y subfamily which activation induces [Ca(2+)](i) and [Na(+)](i) rise and Na(+)-dependent plasma membrane depolarization leading to estradiol secretion.     

Calcium exerts a larger regulatory effect on potassium+ channels in small mesenteric artery mycoytes from spontaneously hypertensive rats compared to Wistar-Kyoto rats

Hypertension is associated with a remodeling of arterial smooth muscle K(+) channels with Ca(2+)-gated K(+) channel (BK(Ca)) activity being enhanced and voltage-gated K(+) channel (K(v)) activity depressed. Because both of these channel types are modulated by intracellular Ca(2+), we tested the hypothesis that Ca(2+) had a larger effect on both BK(Ca) and K(v) channels in arterial myocytes from hypertensive animals. Myocytes were enzymatically dispersed from small mesenteric arteries (SMA) of 12-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Using whole cell patch clamp methods, BK(Ca) and K(v) current components were determined as iberiotoxin-sensitive and -insensitive currents, respectively. The effects of Ca(2+) on these K(+) current components were determined from measurements made with 0.2 and 2 mmol/L external Ca(2+). Increasing external Ca(2+) from 0.2 to 2 mmol/L Ca(2+) increased BK(Ca) currents recorded using myocytes from both WKY rats and SHR with a larger effect in SHR. Increasing external Ca(2+) decreased K(v) currents recorded using myocytes from both WKY and SHR also with a larger effect in SHR. In other experiments, currents through voltage-gated Ca(2+) channels (Ca(v)) measured at 0.2 mmol/L external Ca(2+) were 12 +/- 2% (n = 12) of those recorded at 2 mmol/L Ca(2+) with no differences in percent effect between WKY and SHR. In isolated SMA segments, isometric force development in response to 140 mmol/L KCl at 0.2 mmol/L external Ca(2+) was about 23 +/- 6% (n = 8) of that measured at 2 mmol/L external Ca(2+). These results suggest that an increase in Ca(2+) influx through Ca(v) or in intracellular Ca(2+) secondary to an increase in external Ca(2+) augments BK(Ca) currents and inhibits K(v) currents in SMA myocytes with a larger effect in SHR compared to WKY. This mechanism may contribute to the functional remodeling of K(+) currents of arterial myocytes in hypertensive animals.     

Platelet-derived growth factor (PDGF)-BB produces NO-mediated relaxation and PDGF receptor β-dependent tonic contraction in murine iliac lymph vessels

We studied the effects of PDGF-BB on changes in the diameters of murine lymph vessels with or without intact endothelium. PDGF-BB induced dilation of the lymph vessels with endothelium. Pretreatment with l-NAME or removal of the endothelium caused a significant attenuation in the PDGF-BB-induced dilation. PDGF-BB also produced dose-related reduction of the diameters of the lymph vessels without endothelium. To evaluate intracellular signal transduction and Ca(2+) -dependence of the PDGF-BB-induced tonic contraction, we investigated the effects of imatinib, GW5074 (an inhibitor of Raf-1 kinase), U-73122 (an inhibitor of phospholipase C), and xestospongin C on the PDGF-BB-induced reduction responses. All of these inhibitors caused a significant attenuation in the PDGF-BB-induced reduction response that was significantly decreased by treatment with Ca(2+) -free Krebs-bicarbonate solution or nifedipine. Higher concentrations of PDGF-BB produced a marked reduction of lymph vessel diameter within both high K(+) Krebs-bicarbonate solution and Ca(2+) -free high K(+) Krebs solution containing 1mM EGTA. These findings suggest that PDGF-BB induced endothelium-dependent NO-mediated relaxation of lymphatic smooth muscles in murine lymph vessels. PDGF receptor β-mediated tonic contraction of the muscles through increased Ca(2+) influx through the membrane and the release of membrane-bound and intracellular Ca(2+) .     

Store-operated calcium influx and stimulation of steroidogenesis in rat Leydig cells: role of Ca(2+)-activated K(+) channels

This study evaluates the role of internal calcium store depletion in the activation of ionic fluxes and steroidogenesis in adult rat Leydig cells. Thapsigargin and cyclopiazonic acid, two inhibitors of Ca(2+)-adenosine triphosphatase of internal Ca(2+) stores induced a dose-dependent rise in intracellular Ca(2+) concentrations following kinetics that would not be expected if the calcium rise was dependent only on internal calcium store depletion, but it was in keeping with the presence of calcium influx from the external medium. In fact, chelation of external calcium with EGTA during the plateau phase reduced the intracellular calcium concentration to basal levels. When added in calcium-free medium, thapsigargin and cyclopiazonic acid still induced a rise in the intracellular calcium concentration that was transient, and when calcium was added back to the medium, a rapid and sustained intracellular calcium increase was observed. Thapsigargin and cyclopiazonic acid induced a dose-dependent rise in testosterone secretion in the presence and absence of calcium in the external medium, although in calcium-free medium this stimulatory effect was lower. Leydig cell plasma membrane potential monitoring demonstrated that thapsigargin and cyclopiazonic acid induced first a rapid hyperpolarization, followed by a sustained depolarization phase that was reversed by the addition of the calcium-chelating agent EGTA. In the absence of calcium in the external medium the first phase of hyperpolarization was still present, but it was not followed by plasma membrane depolarization but by the slow return of plasma membrane potential to resting levels. The readdition of calcium to the external medium induced the rapid plasma membrane depolarization. Plasma membrane hyperpolarization was completely abolished by Leydig cell preincubation with the K(+) channel blockers tetraethylammonium and charybdotoxin. Leydig cell preincubation with K(+) channel inhibitors reduced the thapsigargin-stimulated Ca(2+) influx from the external medium and testosterone secretion. These results suggest that internal Ca(2+) stores depletion in rat Leydig cells induces a rise in intracellular Ca(2+), determining important plasma membrane potential variations that influence testosterone secretion.     

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity, although the activity was lower than that of the wild-type SR Ca(2+)-ATPase. Moreover, this Ca(2+)-sensitive ATPase activity was inhibited by ouabain. The chimera NCC, in which Met1-Gly354 of the SR Ca(2+)-ATPase were replaced with the corresponding portion of the Na+,K(+)-ATPase, lost the thapsigargin-sensitive Ca(2+)-ATPase activity seen in CCC and [n/c]CC. [3H]Ouabain binding to [n/c]CC and NCC demonstrated that the affinity for this inhibitor seen in the wild-type chicken Na+,K(+)-ATPase was restored in these chimeric molecules. Thus, the ouabain-binding domains are distinct from the thapsigargin sites; ouabain binds to the amino-terminal portion (Met1 to Asp200) of the Na+,K(+)-ATPase alpha 1 subunit, whereas thapsigargin interacts with the regions after Asp162 of the Ca(2+)-ATPase. Moreover, the amino-terminal 200 amino acids of the Na+,K(+)-ATPase alpha 1 subunit are sufficient to exert ouabain-dependent inhibition even after incorporation into the corresponding portion of the Ca(2+)-ATPase, and the segment Ile163 to Gly354 of the SR Ca(2+)-ATPase is critical for thapsigargin- and Ca(2+)-sensitive ATPase activity.     

Intracellular sodium during ischemia and calcium-free perfusion: a 23Na NMR study

Accumulation of sodium-ions (Na+) in myocardial cells during both ischemia and calcium (Ca2+)-free perfusion has been suggested to play an important role in the damage occurring during subsequent reperfusion and calcium repletion, respectively. We have used 23Na NMR spectroscopy in combination with shift reagents to determine intracellular Na(+)-concentration [( Na+]i) in isolated rat hearts during either control perfusion followed by ischemia and reperfusion, or during control perfusion, Ca(2+)-free perfusion and subsequent ischemia. [Na+]i during control perfusion was found to be 10.5 +/- 0.6 mmol/l. During 30 min of ischemia [Na+]i rose substantially to 25.0 +/- 3.2 mmol/l. During 15 min of reperfusion [Na+]i initially decreased, but leveled off after approximately 3 min and was 17.9 +/- 3.7 mmol/l at the end of the reperfusion period. Most surprisingly, however, no significant increase of [Na+]i was observed during 30 min of Ca(2+)-free perfusion, although severe calcium paradox damage was shown to occur under the used conditions, when calcium was readmitted to the heart. The absence of a rise of [Na+]i during Ca(2+)-free perfusion was substantiated when during subsequent ischemia a similar rise of [Na+]i was observed as during ischemia without previous Ca(2+)-depletion. We conclude that an increased [Na+]i during Ca(2+)-depletion is not a prerequisite for the calcium paradox to occur, but that increased [Na+]i during ischemia may influence the subsequent reperfusion damage through Na(+)-Ca2+ exchange.     

Oleic acid glucose-independently stimulates glucagon secretion by increasing cytoplasmic Ca2+ via endoplasmic reticulum Ca2+ release and Ca2+ influx in the rat islet alpha-cells

The effect of long-chain free fatty acids on glucagon secretion from islet alpha-cells has been a controversial issue. This study examined direct effects of oleic acid (OA) on glucagon release from rat pancreatic islets and on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in single alpha-cells by fura-2 fluorescence imaging. OA at 30 microM increased glucagon release from isolated islets in the presence of low (2.8 mM) and elevated (8.3 mM) glucose concentrations. OA at 6-10 microm concentration-dependently increased [Ca(2+)](i) in alpha-cells, irrespective of glucose concentrations (1.4, 2.8, and 8.3 mM). OA at 10 mum increased [Ca(2+)](i) in 90% of alpha-cells. OA-induced [Ca(2+)](i) increases were strongly inhibited by the endoplasmic reticulum Ca(2+)-pump inhibitors cyclopiazonic acid and thapsigargin and by 2-aminoethoxydiphenyl borate, the blocker of both inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) channels. Furthermore, the amplitude, but not incidence, of OA-induced [Ca(2+)](i) increases was reduced substantially by Ca(2+)-free conditions and mildly by an L-type Ca(2+) channel blocker, nitrendipine, and an ATP-sensitive K(+) channel activator, diazoxide. OA-induced glucagon release was also inhibited mildly by nitrendipine and strongly by 2-aminoethoxydiphenyl borate. These results indicate that OA glucose-independently stimulates glucagon release by increasing [Ca(2+)](i) in rat pancreatic alpha-cells and that the [Ca(2+)](i) increase is triggered by Ca(2+) release from endoplasmic reticulum and amplified by Ca(2+) influx possibly via store-operated channels and via voltage-dependent L-type Ca(2+) channels. The glucose-independent action of OA to stimulate glucagon release from alpha-cells may operate under hypoglycemic conditions when plasma free fatty acids levels are elevated, possibly playing a role in maintaining glucose metabolism.     

Inhibition of protein synthesis sequentially impairs distinct steps of stimulus-secretion coupling in pancreatic beta cells

Proteins with a short half-life are potential sites of pancreatic ss cell dysfunction under pathophysiological conditions. In this study, mouse islets were used to establish which step in the regulation of insulin secretion is most sensitive to inhibition of protein synthesis by 10 microM cycloheximide (CHX). Although islet protein synthesis was inhibited approximately 95% after 1 h, the inhibition of insulin secretion was delayed and progressive. After long (18-20 h) CHX-treatment, the strong (80%) inhibition of glucose-, tolbutamide-, and K(+)-induced insulin secretion was not due to lower insulin stores, to any marked impairment of glucose metabolism or to altered function of K(+)-ATP channels (total K(+)-ATP currents were however decreased). It was partly caused by a decreased Ca(2+) influx (whole-cell Ca(2+) current) resulting in a smaller rise in cytosolic Ca(2+) ([Ca(2+)](i)). The situation was very different after short (2-5 h) CHX-treatment. Insulin secretion was 50-60% inhibited although islet glucose metabolism was unaffected and stimulus-induced [Ca(2+)](i) rise was not (2 h) or only marginally (5 h) decreased. The efficiency of Ca(2+) on secretion was thus impaired. The inhibition of insulin secretion by 15 h of CHX treatment was more slowly reversible (>4 h) than that of protein synthesis. This reversibility of secretion was largely attributable to recovery of a normal Ca(2+) efficiency. In conclusion, inhibition of protein synthesis in islets inhibits insulin secretion in two stages: a rapid decrease in the efficiency of Ca(2+) on exocytosis, followed by a decrease in the Ca(2+) signal mediated by a slower loss of functional Ca(2+) channels. Glucose metabolism and the regulation of K(+)-ATP channels are more resistant. Proteins with a short half-life appear to be important to ensure optimal Ca(2+) effects on exocytosis, and are the potential Achille's heel of stimulus-secretion coupling.     

Extracellular terbium and divalent cation effects on the red blood cell Na pump and chrysoidine effects on the renal Na pump

We examined the effect of extracellular terbium (Tb(3+)) and divalent metal cations (Ca(2+), Sr(2+), and Ba(2+)) on (86)Rb(+) influx into rabbit and human red blood cells. We found that Tb(3+) at 15 and 25 microM was a non-competitive inhibitor of (86)Rb(+) influx suggesting that Tb(3+) is not binding to the transport site. This result reduces the usefulness of Tb(3+) as a potential probe for the E(out) conformation (the conformation with the transport site facing extracellularly). Ba(2+), Sr(2+) and Ca(2+), at concentrations >50 mM, had minimal effects on Rb(+) influx into red blood cells (1 mM Rb-out). This suggests that the outside transport site is very specific for monovalent cations over divalent cations, in contrast to the inside transport site. We also found that chrysoidine (4-phenylazo-m-phenylenediamine) competes with Na(+) for ATPase activity and K(+) for pNPPase activity suggesting it is binding to the E(in) conformation. Chrysoidine and similar compounds may be useful as optical probes of the E(in) conformation.     

Inversion of neurovascular coupling by subarachnoid blood depends on large-conductance Ca2+-activated K+ (BK) channels

The cellular events that cause ischemic neurological damage following aneurysmal subarachnoid hemorrhage (SAH) have remained elusive. We report that subarachnoid blood profoundly impacts communication within the neurovascular unit-neurons, astrocytes, and arterioles-causing inversion of neurovascular coupling. Elevation of astrocytic endfoot Ca(2+) to ～400 nM by neuronal stimulation or to ～300 nM by Ca(2+) uncaging dilated parenchymal arterioles in control brain slices but caused vasoconstriction in post-SAH brain slices. Inhibition of K(+) efflux via astrocytic endfoot large-conductance Ca(2+)-activated K(+) (BK) channels prevented both neurally evoked vasodilation (control) and vasoconstriction (SAH). Consistent with the dual vasodilator/vasoconstrictor action of extracellular K(+) ([K(+)](o)), [K(+)](o) <10 mM dilated and [K(+)](o) >20 mM constricted isolated brain cortex parenchymal arterioles with or without SAH. Notably, elevation of external K(+) to 10 mM caused vasodilation in brain slices from control animals but caused a modest constriction in brain slices from SAH model rats; this latter effect was reversed by BK channel inhibition, which restored K(+)-induced dilations. Importantly, the amplitude of spontaneous astrocytic Ca(2+) oscillations was increased after SAH, with peak Ca(2+) reaching ～490 nM. Our data support a model in which SAH increases the amplitude of spontaneous astrocytic Ca(2+) oscillations sufficiently to activate endfoot BK channels and elevate [K(+)](o) in the restricted perivascular space. Abnormally elevated basal [K(+)](o) combined with further K(+) efflux stimulated by neuronal activity elevates [K(+)](o) above the dilation/constriction threshold, switching the polarity of arteriolar responses to vasoconstriction. Inversion of neurovascular coupling may contribute to the decreased cerebral blood flow and development of neurological deficits that commonly follow SAH.