Mechanisms of delta-hexachlorocyclohexane toxicity: I. Relationship between altered ventricular myocyte contractility and ryanodine receptor function

Several isomers of hexachlorocyclohexanes (HCHs) have been shown to be toxic to mammals. Previous studies have revealed that the delta isomer (delta-HCH) was particularly potent toward disrupting Ca2+ homeostasis in a variety of excitable and nonexcitable cells and altering contractility of cardiac muscle. The effects of the delta and gamma isomers of HCH were further investigated on isolated ventricular myocytes from guinea pig and on single cardiac ryanodine receptor (RyR2) Ca2+-release channels from cardiac SR vesicles. Intracellular Ca2+ transients were examined in electrically stimulated cells using the fluorescent dye indo-1, and twitch contractions of myocytes were analyzed using a video-based edge motion detection system. Exposure of myocytes to delta- but not gamma-HCH depressed the peak of intracellular Ca2+ transients and prolonged recovery time. These effects were correlated with the ability of delta-HCH to inhibit the binding of [3H]ryanodine, a conformationally sensitive probe for RyR2 function, to SR preparations (IC50 = 2 and 18 microM for high- and low-affinity interactions, respectively). Measurements of single-channel gating kinetics under voltage-clamp provided direct evidence of a potent isoform-selective activation of RyR2 by delta-HCH. Results from these studies revealed that delta-HCH alters Ca2+ homeostasis and contractility in cardiac myocytes and that the mechanism can be ascribed, at least in part, to a direct interaction with the RyR2 channel complex.     

Comparison of [3H]ryanodine receptors and Ca++ release from rat cardiac and rabbit skeletal muscle sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) vesicles prepared from rat ventricle muscle are isolated, and their [3H]ryanodine-binding and calcium transport properties are studied in detail under active loading conditions in the presence of pyrophosphate. Experiments are performed in tandem with rabbit skeletal SR under identical conditions to allow direct comparisons of the mechanisms by which activators and inhibitors influence the calcium release channel. Ca(++)-induced Ca++ release is demonstrated with both preparations and the cardiac channel is about 1.5-fold more sensitive to activation by Ca++, which is in excellent quantitative agreement with the ability of Ca++ to activate [3H]ryanodine-binding sites. The cardiac and skeletal receptors show major quantitative differences with respect to sensitivity to pharmacologic modulators, cations and pH. The inhibitors ruthenium red, Mg++ and neomycin are significantly more potent in inhibiting the skeletal receptor, whereas the activators daunorubicin and caffeine are significantly more potent towards the cardiac receptor. The ATP analog, beta,gamma-methyleneadenosine 5'-triphosphate, enhances the binding of [3H]ryanodine to the high-affinity site in skeletal SR by a factor of 4 but has a negligible effect on the cardiac receptor, although at suboptimal Ca++ for the binding of ryanodine, beta,gamma-methyleneadenosine 5'-triphosphate activates the cardiac receptor to a greater extent. High levels of salt (1 M NaCl) enhance the rate of [3H]ryanodine association with its binding sites in both preparations, although they selectively reduce the binding-site capacity in skeletal SR due to a failure to maintain a stable equilibrium. Although high- and low-affinity binding of [3H]ryanodine have a similar response to changing pH, the skeletal receptors are significantly more sensitive to pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-methyl-D-aspartic acid (NMDA) and non-NMDA receptors regulating hippocampal norepinephrine release. II. Evidence for functional cooperation and for coexistence on the same axon terminal.

The possible interactions between activation of N-methyl-D-aspartic acid (NMDA) receptors and non-NMDA receptors regulating the release of [3H]norepinephrine [( 3H]NE) have been investigated in superfused synaptosomes from rat hippocampus. NMDA--at a concentration (100 microM) which, in a medium containing 1.2 mM Mg++ ions, did not evoke [3H]NE release--acquired releasing activity in the presence of equimolar concentrations of quisqualic acid (QA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. The [3H] NE release evoked by NMDA plus QA in the presence of Mg++ ions was Ca(++)-dependent, partly tetrodotoxin-sensitive, inhibited by clonidine but insensitive to desipramine. The NMDA receptor antagonists D-2-amino-5-phosphonopentanoic acid (D-AP5) and (+)-5-methyl-10,11-dihydro-5-H-dibenzo[a,d]cycloepten-5,10-imine (MK-801) antagonized the NMDA-induced [3H]NE release in Mg(++)-free medium; the IC50 values amounted, respectively, to 81.4 microM and to 1.11 microM. When NMDA was tested in the presence of QA and Mg++ ions, the affinity of D-AP5 was enormously increased (IC50 = 40 nM; i.e., more than 6 orders of magnitude); the affinity of MK-801 was found to be augmented by 350-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

9-methyl-7-bromoeudistomin D, a powerful radio-labelable Ca++ releaser having caffeine-like properties, acts on Ca(++)-induced Ca++ release channels of sarcoplasmic reticulum.

9-Methyl-7-bromoeudistomin D (MBED), a derivative of eudistomin D isolated from a marine tunicate, induced Ca++ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) in the same way as that of caffeine, followed by spontaneous Ca++ reuptake in the Ca++ electrode experiment. The rate of 45Ca++ efflux from HSR vesicles was accelerated markedly by MBED or caffeine in a concentration-dependent manner. The 50% effective concentrations of MBED and caffeine were approximately 1 microM and 1 mM, respectively, indicating that MBED is 1000 times more potent than caffeine in HSR. Procaine, ruthenium red or Mg++ caused concentration-dependent inhibition of MBED-triggered Ca++ release from HSR. The bell-shaped profile of Ca++ dependence for MBED is very similar to that of caffeine. The caffeine-produced maximum response of 45Ca++ efflux was increased further by adenosine-5'-(beta, gamma-methyl-ene)triphosphate, whereas that was not changed by MBED. MBED also caused Ca++ release from sarcoplasmic reticulum (SR) of chemically skinned fibers. These stimulatory effects of MBED on the Ca++ release from skeletal muscle SR were almost indistinguishable from those of caffeine except the difference in potencies. The [3H]ryanodine binding to junctional terminal cisternae membranes was not inhibited by MBED or caffeine. MBED did not cause Ca++ release from the light fraction of fragmented SR and turbidity change of mitochondrial suspension. These observations suggest a most likely idea that MBED binds to the caffeine-binding site in the Ca channel protein and thus produces the potentiation of Ca(++)-induced Ca++ release from SR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impairment of the ryanodine-sensitive calcium release channels in the cardiac sarcoplasmic reticulum and its underlying mechanism during the hypodynamic phase of sepsis.

Changes in Ca2+-induced Ca2+ release in cardiac sarcoplasmic reticulum (SR) during different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). The 45Ca2+ release studies show that the amount of Ca2+ released from the passively and the actively loaded SR vesicles was unaffected during the early sepsis (9 h after CLP), but it was significantly decreased during the late phase (18 h after CLP) of sepsis. The [3H]ryanodine binding assays reveal that the Bmax for ryanodine binding was unaffected during the early phase, but was decreased by 32.1% during the late phase of sepsis. The affinity of ryanodine receptor for Ca2+ remained unchanged during sepsis. ATP, AMP-PCP, and caffeine stimulated binding, while MgCl2 and ruthenium red inhibited [3H]ryanodine binding in control, early sepsis, and late sepsis groups. The EC50 and IC50 values for these regulators were unaffected during the progression of sepsis. Digestion of control SR with phospholipase A2 decreased [3H]ryanodine binding and the decrease was reversible by the addition of phosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylserine (PS). Addition of PC, PE, or PS to the SR isolated from septic rats stimulated [3H]ryanodine binding. These data demonstrate that Ca2+-induced Ca2+ release from cardiac SR remained relatively unaffected during the early phase, but was significantly impaired during the late phase of sepsis. The sepsis-induced impairment in SR Ca2+ release is a result of a quantitative reduction in the number of Ca2+ release channels. Furthermore, the reduction is associated with a mechanism involving a modification of membrane lipid profile in response to certain stimuli such as activation of phospholipase A2.     

Alpha-2 adrenergic inhibition of Ca(++)-evoked [3H]norepinephrine release from synaptosomes is blocked by depolarization

The Ca(++)-evoked release of [3H]norepinephrine was used in these studies to investigate presynaptic regulation of norepinephrine release. In hippocampal synaptosomes, previously unexposed to Ca++ during isolation and superfusion, 1.25 mM Ca++ evoked a modest (4 to 7% of total stores) release of [3H]norepinephrine with 4.5 mM [K+] present. The alpha-2 adrenergic agonist clonidine inhibited 60% of the Ca(++)-evoked [3H]norepinephrine release. The alpha-2 adrenergic antagonists idazoxan and yohimbine reversed clonidine inhibition of release whereas the alpha-1 antagonist prazosin did not. Increasing the [K+] before Ca++ exposure increased [3H]norepinephrine release, and at 20 [K+] the release increased to over 20% of total stores. However, at [K+] above 9 mM, inhibition of Ca(++)-evoked release by clonidine decreased, and by 20 mM [K+] clonidine no longer inhibited release. Release was unaffected by 5 microM idazoxan or the opiate antagonist naloxone at 15 or 20 mM [K+]. The K+ channel blockers tetraethylammonium (5 mM) and 4-aminopyridine (0.1 mM) increased Ca(++)-evoked release almost 4-fold above control (4.5 mM [K+] present). Neither clonidine nor idazoxan affected Ca(++)-evoked release with the K+ channel blockers present. Therefore, even though K+ channel blockers and 20 mM [K+] increase neurotransmitter release, it is not autoreceptor activation by released endogenous norepinephrine that is responsible for blocking alpha-2 inhibition, but the depolarization produced by these treatments. The 20 mM [K+] blockade of alpha-2 inhibition was decreased by lowering the [Ca++] in the superfusion buffer. Therefore, synaptosomal accumulation of Ca++ may partially explain the loss of alpha-2 inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for intraluminal Ca++ regulatory site defect in sarcoplasmic reticulum from malignant hyperthermia pig muscle.

Malignant hyperthermia (MH) is a pharmacogenetic disease of humans and various animal species that predisposes to a life-threatening, anesthetic agent-induced syndrome. MH is thought to be a consequence of abnormal, sustained increases in myoplasmic Ca++ and sarcoplasmic reticulum (SR) membranes from MH muscle have been shown to have a Ca++ release channel defect. In the present study we have tested a hypothesis that the abnormal Ca++ release mechanism in MH can be expressed when Ca++ is loaded in the presence of pyrophosphate. SR membrane vesicles isolated from normal and MH pig muscle were loaded with Ca++ in the presence and absence of pyrophosphate until Ca(++)-induced Ca++ release occurred. Under both circumstances the threshold amount of Ca++ loaded until Ca++ release occurred was lower in the SR from MH pig skeletal muscle. This difference in amount of Ca++ preload is not explained by results obtained comparing rates of Ca++ uptake, number of ryanodine binding sites or the amounts of calsequestrin among SR vesicles from MH and normal muscle. We conclude from this study that use of pyrophosphate for Ca++ loading does not ablate the abnormal Ca++ release in SR from MH muscle, suggesting the study can be done on small amounts of SR from biopsied human muscle. The data also suggest that abnormality in an intraluminal, low affinity Ca++ binding site regulating Ca++ release occurs in the SR membrane of MH pig muscle.     

Mechanism of vascular smooth muscle contraction by sodium fluoride in the isolated aorta of rat and rabbit.

The purpose of this study was to determine the cellular basis for fluoride ion (F-)-induced contractions of isolated aortic rings from both the rat and the rabbit. The F- contractions were not affected by endothelial denudation but were enhanced in the presence of A (0.1 or 1.0 mM) added to the bathing Krebs' solution. The contractile effect of F- also was not modified by bathing with Ca(++)-free + ethylene glycol bis(b-aminoethylether)-N,N-tetracetic acid Krebs' solution or nifedipine (10 microM), but was attenuated by inorganic (Cd++, Co++ and Ni++) Ca++ antagonists in normal and Ca(++)-free Krebs' media. Bis(o-aminophenoxy)-ethane-N-N-N'-N'-tetraacetic acid, ryanodine and intracellular Ca++ modulators, respectively, caused 36.1 +/- 6.1%, 16.4 +/- 6.8% and 52.3 +/- 7.3% inhibition of the contractile response to F- in a Ca(++)-free media while causing near complete inhibition of norepinephrine-induced contractions. F- contractions were also inhibited by the calmodulin antagonists W-7 and calmidazolium (IC50 = 23.0 +/- 7.0 and 45.0 +/- 10.0 microM, respectively). On the other hand, the protein kinase C antagonists staurosporine and H-7 potently (IC50 = 0.016 +/- 0.007 and 1.1 +/- 0.5 microM, respectively) inhibited the fluoride-induced contractions. Aortic rings from the rabbit were similarly potently antagonized by the protein kinase C inhibitors, however, K(+)-induced contractions were also equally sensitive to these agents in both rat and rabbit tissues. The putative phospholipase C inhibitor neomycin was significantly less effective (IC50 = 13.0 +/- 5.0, 0.44 +/- 0.09 and 0.89 +/- 0.40 mM) at inhibiting F- than norepinephrine and KCl contractile effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of K+ channel-dependent as well as -independent components of pinacidil-induced vasodilation

The mechanisms of pinacidil-induced direct vasodilation were studied in vitro in RMA and RAO. In RMA, pinacidil produced dose-dependent relaxations of norepinephrine (5 microM)-induced contractions with an IC50 of 0.2 microM. This component of pinacidil relaxation appeared to be dependent on K+ conductance because pretreatment with tetraethylammonium (10 mM), Ba++ (0.5 mM), glyburide (1 microM) and 20 mM K+ all caused a rightward shift of the pinacidil dose-response curve (DRC) and a corresponding increase in the pinacidil IC50. However, additional relaxation effects of pinacidil were still evident in the presence of various K+ channel blockers. Pinacidil also showed a relaxation DRC under the condition of 80 mM K+ contraction in both RMA and RAO with IC50 values of 27 and 50 microM, respectively. Pinacidil could also produce maximal relaxation in RMA and RAO remained unaffected in 145 mM K+ (zero Na+) depolarizing solution suggesting a lack of dependence on Na(+)-Ca++ exchange mechanism for this action of pinacidil. Studies using 1 or 3 min pulse labeling with 45Ca showed an absence of an inhibitory effect of pinacidil (at 50 and 100 microM) on unidirectional 45Ca influx stimulated by high-K+. Net 45Ca uptake studies showed that pinacidil inhibited high-K+ stimulated 45Ca uptake at 100 but not at 50 microM. Ryanodine (10-100 microM) was used as a tool to investigate the role of sarcoplasmic reticulum (SR) in this action of pinacidil. Under the condition in which ryanodine (10-100 microM) treatment was found to cause the SR to be nonfunctional, pinacidil relaxation DRC remained unaltered, suggesting a lack of a stimulatory effect of pinacidil on SR Ca++ accumulation. These data thus show that the K+ channel-independent effect of pinacidil does not involve to any significant degree an effect of pinacidil on plasmalemmal voltage-sensitive Ca++ channels, SR Ca++ stores, Na(+)-Ca++ exchange or membrane hyperpolarization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tectoridins modulate skeletal and cardiac muscle sarcoplasmic reticulum calcium-release channels

The isoflavones tectoridin (TTR) and 3'-hydroxy TTR (3'-TTR) were isolated from an Ayurvedic herbal preparation Vac? and evaluated for their affinity and effect on ryanodine receptors (RyR) using junctional sarcoplasmic reticulum vesicles (JSRVs). In [(3)H]ryanodine displacement binding affinity assays, TTR and 3'-TTR exhibited IC(50) values of 17.3 +/- 1.3 microM (K(d) = 6.7 +/- 0.4 microM) and 6.6 +/- 1.4 microM (K(d) = 2.4 +/- 0.2 microM), respectively, for fast skeletal muscle RyR (RyR1) compared with an IC(50) value for ryanodine of 6.2 +/- 0.4 nM (K(d) = 2.4 nM). TTR demonstrated a 3-fold higher affinity for cardiac RyR (RyR2) [IC(50) value of 5.2 +/- 0.6 microM (K(d) = 0.95 +/- 0.3 microM)] than for RyR1. The displacement isotherms for both TTRs paralleled that for ryanodine, consistent with the notion that all three are likely binding to similar site(s) on the receptors. Calcium efflux from and calcium influx into JSRVs were used to measure function effects of TTRs on binding to RyR. In calcium efflux assays, TTR (up to 1 mM) enhanced the release of (45)Ca(2+) from JSRVs in a concentration-dependent manner (EC(50act) of 750 microM). Higher concentrations deactivated (partially closed) RyR1. 3'-TTR had similar effects, but was approximately 2-fold more potent, exhibiting an EC(50act) value of 480 microM. Using passive calcium influx assays, TTR activated and deactivated RyR1 in a time- and concentration-dependent manner. The aglycone tectorigenin also was effective in displacing [(3)H]ryanodine from RyR1 but not from RyR2. These results demonstrate that TTRs are capable of interacting at ryanodine binding sites to differentially modulate fast skeletal and cardiac calcium-release channels.     

Effects of Ca++ on [3H]diltiazem binding and its allosteric interaction with dihydropyridine calcium channel binding sites in the rat cortex

In membranes from the rat cerebral cortex, the benzothiazepine [3H]diltiazem and the dihydropyridine [3H]nitrendipine label distinct but allosterically interacting calcium channel antagonist recognition sites. In the present study we evaluated the relationship between Ca++ and calcium channel antagonists binding sites within the voltage-dependent Ca++ channel. [3H]Nitrendipine binding to the rat cerebral cortex at 37 degrees C is not inhibited by Ca++, studied as CaCl2, in concentrations up to 10 mM. In contrast, [3H]diltiazem binding under these conditions is inhibited by Ca++ with an IC50 of 0.31 mM. The inhibition of diltiazem binding by Ca++ is reflected in an increase in the EC50 of diltiazem for enhancement of [3H]nitrendipine binding at 37 degrees C but not in the magnitude of its maximal heterotropic positive cooperative effect. Similarly, Ca++ increases the IC50 of verapamil for inhibition of [3H]nitrendipine binding at 37 degrees C without affecting the magnitude of its heterotropic negative cooperative effect. Schild analyses of these data indicate that Ca++ is an essentially competitive antagonist of the allosteric effects of diltiazem (KB = 0.32 mM) and verapamil (KB = 0.33 mM). At 0 degrees C, Ca++ is a negative allosteric inhibitor of the [3H]nitrendipine binding site (IC50 = 0.1 mM) and selectively increases the IC50 of diltiazem and verapamil for the inhibition of [3H]nitrendipine binding to membranes from the rat cerebral cortex. It may thus be suggested that the diltiazem and verapamil recognition sites are closely linked or identical with Ca++ binding site within the slow voltage-dependent Ca++ channel.     

Local anesthetics differentiate dihydropyridine calcium antagonist binding sites in rat brain and cardiac membranes

Local anesthetics were used to probe differences in the binding of [3H]nitrendipine to dihydropyridine calcium antagonist binding sites on rat brain and cardiac membranes. Local anesthetics inhibited [3H]nitrendipine binding to brain and cardiac membranes with the rank order of potency, dibucaine = proadifen much greater than tetracaine greater than meproadifen greater than RAC-109 (S) greater than RAC-109 (R) greater than benzocaine. Lidocaine, procaine, piperocaine and bupivacaine produced either a small potentiation or inhibition of [3H]nitrendipine binding. Dibucaine inhibited [3H]nitrendipine binding to brain membranes (IC50, 4.9 +/- 0.5 microM) by increasing the Kd, whereas in cardiac membranes (IC50, 8.5 +/- 0.9 microM) it both increased the Kd and decreased the maximum binding site capacity of [3H]nitrendipine. The potency of dibucaine to inhibit [3H]nitrendipine binding was reduced in both tissues by monovalent (Li+ greater than Na+ = K+ = Rb+; EC50, 40-50 mM) and divalent (Ca++, Mg++ and Mn++; EC50, 10-50 microM) cations. These cations reduced the effect of dibucaine on the Kd of [3H]nitrendipine in brain and on the maximum binding site capacity of [3H]nitrendipine in cardiac membranes. Inhibition of [3H]nitrendipine binding by dibucaine was best described by high (2 microM) and low (50 microM) affinity sites. The apparent affinities of these sites, but not the fractional occupancies, were similar in brain and cardiac membranes. Na+ modulated the occupancies of these sites in brain, but not in cardiac membranes, whereas Ca++ inhibited occupancy of the high affinity site in both tissues. The effects of Li+ were similar to those of Ca++. These findings indicate that brain and cardiac dihydropyridine calcium antagonist binding sites are coupled to different allosteric effectors or exist in a different membrane environment.     

Membrane events and ionic processes involved in dopamine release from tuberoinfundibular neurons. I. Effect of the inhibition of the Na+,K+-adenosine triphosphatase pump by ouabain

In the present study we investigated the membrane events and the ionic processes which mediate the stimulatory effect of ouabain on the release of endogenous dopamine (DA) and "previously taken-up" [3H]DA release from rat hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons. Ouabain (0.1-1 mM) dose-dependently stimulated endogenous DA and "newly taken-up" [3H]DA release. This effect was counteracted partially by nomifensine (10 microM). Removal of Ca++ ions from the extracellular space in the presence of the Ca++-chelator ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid prevented completely ouabain-elicited [3H]DA release. Lanthanum (1 mM) and cobalt (2 mM), two inorganic Ca++-entry blockers, were able to inhibit this stimulatory effect, whereas verapamil (10 microM) and nitrendipine (50 microM), two organic antagonists of the voltage-operated channel for Ca++ ions, failed to affect ouabain-induced [3H]DA release. By contrast, adriamycin (100-300 microM), a putative inhibitor of cardiac Na+-Ca++ antiporter, dose-dependently prevented ouabain-induced [3H]DA release from TIDA neurons. Finally, tetrodotoxin reduced digitalis-stimulated [3H]DA release. In conclusion, these results seem to be compatible with the idea that the inhibition of Na+,K+-adenosine triphosphatase by ouabain stimulates the release of [3H]DA from a central neuronal system like the TIDA tract and that this effect is critically dependent on the entrance of Ca++ ions into the nerve terminals of these neurons. In addition the Na+-Ca++ exchange antiporter appears to be the membrane system which transports Ca++ ions into the neuronal cytoplasm during Na+,K+-adenosine triphosphatase inhibition. The enhanced intracellular Ca++ availability triggers DA release which could occur partially through a carrier-dependent process.     

Mechanisms of delta-hexachlorocyclohexane toxicity: II. Evidence for Ca2+-dependent K+-selective ionophore activity

delta-Hexachlorocyclohexane (delta-HCH) interacts with cardiac ryanodine-sensitive Ca2+ channels (RyR2), accounting in part for altered Ca2+ transients and contractility (reported in companion report). Analysis of channel gating kinetics in the presence of delta-HCH also revealed a nonfluctuating membrane current that remained even after RyR2 channels were blocked. We further elucidated the nature of a direct interaction between delta-HCH and biological membranes by measuring ionic currents across planar lipid bilayers made from defined lipids lacking cellular protein using voltage-clamp. Dimethyl sulfoxide, in the presence or absence of 50 microM gamma-HCH (lindane) or delta-HCH, produced negligible steady-state current with symmetric 100 mM CsCl in the range of +/-50 mV. However, the addition of 50 microM Ca2+ to the bilayer chamber in the presence of delta-HCH induced a profound increase in ionic permeability that was not seen in the presence of gamma-HCH or dimethyl sulfoxide control. Significantly, the permeability increase 1) was proportional with increasing Ca2+ to approximately 600 microM and saturated between 1 and 2 mM Ca2+ regardless of holding potential, 2) occurred only when delta-HCH and Ca2+ were added to the same side of the membrane, and 3) was independent of the order of addition or of the side of the membrane to which delta-HCH and Ca2+ was added. The Ca2+-dependent current produced by delta-HCH was highly selective for monovalent cations (K+ > Cs+ > Na+), with negligible conductance for Ca2+ or Cl-. In symmetric 100 mM K+, the conductance induced with 50 microM concentration each of delta-HCH and Ca2+ was 4.25 pA/mV. The results show that delta-HCH increases the ionic permeability of phospholipid membranes by two distinct Ca2+-dependent mechanisms: one mediated through RyR and the other mediated by a unique ionophore activity.     

Inhibition of norepinephrine and acetylcholine release from human neocortex by omega-conotoxin GVIA

Superfused slices of human neocortex, prepared from surgically removed tissue (to gain access to subcortical tumors) and prelabeled selectively with [3H]norepinephrine (NE) or [3H]choline, were stimulated electrically to evoke tritium overflow. This tritium overflow was abolished by the sodium channel blocker tetrodotoxin and by withdrawal of extracellular Ca++. Thus, the action potential-induced, exocytotic tritium overflow supports the assumption of a quasiphysiological release of NE from noradrenergic and of acetylcholine (ACh) from cholinergic nerve terminals, respectively. In addition, the modulation of NE release by adrenoceptor ligands displayed the appropriate pharmacology of alpha-2 autoreceptors; ACh release was modulated by muscarinic ligands. Both NE and ACh release decreased with the age of the patients. The effects of drugs on NE and ACh release were not age-related. The peptide modulator of the N-type voltage sensitive Ca++ channel, omega-conotoxin GVIA, inhibited NE release with an IC50 of about 14 nM and ACh release with an IC50 of about 3 nM, whereas L-type modulators were ineffective. The binding of [125I]omega-conotoxin GVIA to human neocortical membranes was of high affinity (KD = 1.3 pM) to one site (nH = 0.97) of substantial density (maximum binding = 878 fmol/mg of protein); the binding of the L-type modulator [3H]isradipine to these membranes was also of high affinity (KD = 89 pM) to one site (nH = 1.03) of lesser density (maximum binding = 429 fmol/mg of protein). In conclusion, Ca++ entry through N-type Ca++ channels, rather then L-type Ca++ channels, predominates in subserving NE and ACh release from noradrenergic and cholinergic nerve terminals, respectively, of human neocortex.     

Mechanism of inhibitory effects of azelastine on smooth muscle contraction.

The mechanism of inhibitory effects of azelastine, an antiallergic and antiasthmatic agent, on depolarization- and alpha-1 adrenergic agonist-induced contractions of intact smooth muscle was studied. The effects of azelastine on membrane currents were determined in isolated guinea pig ileum smooth muscle cells with the whole-cell clamp technique; the effects on contraction were evaluated in receptor- and G-protein-coupled, alpha-toxin-permeabilized rabbit femoral artery and portal vein smooth muscle strips. Azelastine (1-20 microM), like dihydropyridines, inhibited spontaneous rhythmic and high K(+)-induced contractions, mainly through inhibition of the voltage-dependent (L-type) Ca++ current. The tonic component of high K+ contractions was inhibited more than the phasic component, correlating to voltage-dependent inhibition of Ca++ current by the drug. Azelastine (IC50 of 0.25 microM), a known histamine blocker, also reversibly inhibited alpha-1 agonist-induced contractions in the presence and absence of extracellular Ca++. Both major pathways of pharmacomechanical coupling, agonist-induced Ca++ release from the sarcoplasmic reticulum and Ca++ sensitization of the regulatory/contractile apparatus were blocked by the same concentration of drug in permeabilized as in intact muscle. Inositol 1,4,5-trisphosphate-induced Ca++ release and guanosine 5'-O-(tau-thiotriphosphate)-induced Ca++ sensitization, however, were not inhibited. Azelastine at high (greater than 10 microM) concentrations reversibly inhibited Ca(++)-activated contraction, more potently at lower Ca++ concentration and in phasic smooth muscle, but inhibited neither adenosine 5'-O-(tau-thiotriphosphate)-induced, Ca(++)-independent nor phorbol ester-induced contractions. These results indicate that azelastine is a genuine Ca++ antagonist that inhibits voltage-gated Ca++ inward current and agonist-induced Ca++ release and Ca++ sensitization.     

Ryanodine: antithetical calcium channel effects in skeletal muscle sarcoplasmic reticulum

The effects of ryanodine, a neutral alkaloid, on the Ca++ uptake and Ca++ release properties of a skeletal muscle isolated sarcoplasmic reticulum (SR) preparation were evaluated. Ryanodine had no effect on the rate of oxalate-facilitated Ca++ uptake in this SR. Ruthenium red, which reportedly blocks Ca++ channels, increased Ca++ uptake by 2-fold in the SR. Although no effect of ryanodine on Ca++ transport by SR was observed, notable effects on Ca++-induced Ca++ release pathways were discovered. Ryanodine acts on the same Ca++ channels that are affected by ruthenium red. When these Ca++ channels were activated by Ca++ to an open state in the presence of ryanodine, then ryanodine maintained the channel in an activated, open state. However, once Ca++ was taken up by the SR, ryanodine tended to lock the channel in a closed state, producing a condition refractory to Ca++-induced Ca++ release. Thus, dual, opposing effects of ryanodine were demonstrated. In a fast-reaction kinetics measurement of Ca++-induced Ca++ release, both rate and amount of Ca++ release were reduced by ryanodine, and Ca++ appeared to act in a noncompetitive, antagonistic mode. These unusual, antithetical effects of ryanodine on the Ca++ efflux pathway are not mechanistically defined by this study, but they reveal the potential value of ryanodine as a probe for exploring Ca++ channel function.     

Stereoselective secretion of atenolol from PC12 cells

Stereoselective storage and release of the cardioselective beta adrenergic receptor antagonist atenolol was studied using cultured PC12 cells as a neural model. [3H]Atenolol efflux from preloaded PC12 cells was increased 4-fold in response to membrane depolarization by elevated extracellular potassium (50 mM). [3H]Norepinephrine release was enhanced 4.5-fold under the same conditions. Potassium-induced release of both [3H] atenolol and [3H]norepinephrine was inhibited completely in the absence of extracellular calcium. [3H]Atenolol release from PC12 cells was also reduced by the calcium channel antagonist nifedipine (IC50 = 1.6 +/- 0.5 nM). In addition, the calcium channel agonist BAY K8644 (1 microM) significantly enhanced potassium-induced [3H]atenolol efflux. After loading overnight, accumulation and storage of the (-)-enantiomer of atenolol by PC12 cells was found to be approximately 3-fold greater than that of the (+)-enantiomer. The (-)-enantiomer of atenolol was also preferentially released by 50 mM potassium with a (-)/(+)-enantiomer ratio of 3.6 to 1. The results support the existence within neurosecretory cells of storage and calcium-dependent release mechanisms which result in stereoselective secretion of the (-)-or active enantiomer of atenolol in response to membrane depolarization.     

Inhibitory action of peripheral-type benzodiazepines on dopamine release from PC12 pheochromocytoma cells.

Characteristics of the benzodiazepine inhibition of dopamine (DA) release in PC12 cells were investigated. Diazepam inhibited DA release evoked by high concentrations of extracellular K+ in a dose-dependent manner (IC50, 10 microM). Ro 5-4864 [7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine- 2-one], a peripheral-type benzodiazepine, also inhibited DA release effectively. PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinoline carboxamide], a benzodiazepine generally considered a peripheral-type benzodiazepine receptor antagonist, did not antagonize the inhibition induced by diazepam, but rather inhibited DA release itself. On the other hand, the central-type benzodiazepines, clonazepam and Ro 15-1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a] [1,4]benzodiazepine-3-carboxylate) did not affect the DA release. Diazepam, Ro 5-4864 and PK 11195 also inhibited a Ba(++)-current carried by voltage-gated Ca++ channels, and diazepam suppressed an increase in intracellular Ca++ evoked by 80 mM extracellular K+ as measured by the fura-2 method. These results suggest that the inhibitory action of diazepam and other benzodiazepines on DA release from PC12 cells may be mediated through one type of peripheral-type benzodiazepine receptors which are coupled to voltage-gated Ca++ channels and that these receptors may not necessarily be the same as those in other tissues.     

Characteristics of the binding of [3H]nitrendipine to rabbit ventricular membranes: modification by other Ca++ channel antagonists and by the Ca++ channel agonist Bay K 8644

This study was carried out to characterize [3H]nitrendipine binding to cardiac membranes and to test the hypothesis that high affinity binding of Ca++ channel antagonists and agonists is to Ca++ channels. Binding was specific, rapid, reversible and stereoselective. The relative order of potency of nifedipine analogs for inhibition of binding was the same as that for inhibition of smooth and cardiac muscle contraction. Results with diltiazem, verapamil and lidoflazine were consistent with the hypothesis that nondihydropyridine Ca++ channel antagonists act at one or more sites allosterically linked to the 1,4-dihydropyridine site in cardiac cells. The Ca++ channel agonist Bay K 8644 [methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyr idine- 5-carboxylate] displaced specifically bound [3H]nitrendipine in an apparently competitive manner with an IC50 value of 5 nM. The results suggest that organic antagonists do not act by physically blocking the Ca++ channel. The data also support the hypothesis that the high affinity binding sites for [3H]nitrendipine in isolated cardiac membranes are associated with Ca++ channels that are inactivated or are otherwise unavailable for opening.