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Biomedical subjects

J P Reuben

Publications and source records attributed to J P Reuben.

At least 19 recordsLinked to original sources
PubMed2000

Brachial plexus anesthesia with verapamil and/or morphine.

UNLABELLED: Calcium channel blockers potentiate the analgesic properties of both local anesthetics and opioids. We examined the analgesic effects of administering morphine, verapamil, or its combination into the brachial plexus sheath with lidocaine in 75 patients undergoing upper extremity orthopedic surgery. All patients received brachial plexus anesthesia with 40 mL of 1.5% lidocaine and epinephrine 5 microg/mL. In addition, patients were randomized to 1 of 5 groups: Group 1 received IV saline; Group 2 received IV verapamil 2.5 mg and morphine 5 mg; Group 3 received IV verapamil 2.5 mg and morphine 5 mg was added to the lidocaine solution; Group 4 received IV morphine 5 mg and verapamil 2.5 mg was added to the lidocaine solution; and Group 5 received verapamil 2.5 mg and morphine 5 mg were added to the lidocaine solution. Postoperatively, patients rated their pain (0-10) at 1, 6, 12, and 24 h. Patients were instructed to take 1 acetaminophen 325 mg/oxycodone 5 mg tablet every 3 h whenever the pain score exceeded 3. Analgesic duration was significantly increased in those patients receiving brachial plexus blocks with morphine (Groups 3 and 5) (P < 0.005). The total 24 h acetaminophen/oxycodone use was also less in Groups 3 and 5 (P < 0. 03). Duration of anesthesia (time of abolition of pinprick response) was significantly increased in those patients receiving brachial plexus blocks with verapamil (Groups 4 and 5) (P = 0.002). We conclude that the addition of verapamil to brachial plexus block with lidocaine can prolong the duration of sensory anesthesia, but it had no effect on analgesic duration of 24 h analgesic use. IMPLICATIONS: The addition of verapamil to brachial plexus block with lidocaine and morphine prolongs the duration of sensory anesthesia, but has no effect on analgesic duration or 24 h analgesic use.

Adjuvants, Anesthesia↗
PubMed1994

Tremorgenic indole alkaloids potently inhibit smooth muscle high-conductance calcium-activated potassium channels.

Tremorgenic indole alkaloids produce neurological disorders (e.g., staggers syndromes) in ruminants. The mode of action of these fungal mycotoxins is not understood but may be related to their known effects on neurotransmitter release. To determine whether these effects could be due to inhibition of K+ channels, the interaction of various indole diterpenes with high-conductance Ca(2+)-activated K+ (maxi-K) channels was examined. Paspalitrem A, paspalitrem C, aflatrem, penitrem A, and paspalinine inhibit binding of [125I]charybdotoxin (ChTX) to maxi-K channels in bovine aortic smooth muscle sarcolemmal membranes. In contrast, three structurally related compounds, paxilline, verruculogen, and paspalicine, enhanced toxin binding. As predicted from the binding studies, covalent incorporation of [125I]ChTX into the 31-kDa subunit of the maxi-K channel was blocked by compounds that inhibit [125I]ChTX binding and enhanced by compounds that stimulate [125I]ChTX binding. Modulation of [125I]ChTX binding was due to allosteric mechanisms. Despite their different effects on binding of [125I]ChTX to maxi-K channels, all compounds potently inhibited maxi-K channels in electrophysiological experiments. Other types of voltage-dependent or Ca(2+)-activated K+ channels examined were not affected. Chemical modifications of paxilline indicate a defined structure-activity relationship for channel inhibition. Paspalicine, a deshydroxy analog of paspalinine lacking tremorgenic activity, also potently blocked maxi-K channels. Taken together, these data suggest that indole diterpenes are the most potent nonpeptidyl inhibitors of maxi-K channels identified to date. Some of their pharmacological properties could be explained by inhibition of maxi-K channels, although tremorgenicity may be unrelated to channel block.

Allosteric Regulation↗
PubMed1993

An activator of calcium-dependent potassium channels isolated from a medicinal herb.

Large-conductance calcium-dependent potassium (maxi-K) channels play an important role in regulating the tone of airway smooth muscle and the release of bronchoconstrictive substances from nerves in the lung. Crude extracts of Desmodium adscendens, a medicinal herb used in Ghana as a treatment for asthma, inhibit binding of monoiodotyrosine charybdotoxin (125I-ChTX) to receptor sites in bovine tracheal smooth muscle membranes that have been shown to be associated with maxi-K channels. Using this assay, three active components have been purified and identified by NMR and MS. Comparison with authentic samples revealed the three active components as the known triterpenoid glycosides dehydrosoyasaponin I (DHS-I), soyasaponin I, and soyasaponin III. The most potent of these compounds, DHS-I, is a partial inhibitor of 125I-ChTX binding (Ki = 120 nM, 62% maximum inhibition). Inhibition of 125I-ChTX binding is primarily due to a decrease in the observed maximum number of binding sites, with a smaller decrease in affinity. DHS-I increases the rate of toxin dissociation from its receptor, suggesting that modulation of ChTX binding occurs through an allosteric mechanism. DHS-I reversibly increases the open probability of maxi-K channels from bovine tracheal smooth muscle incorporated into planar lipid bilayers when applied to the intracellular, but not the extracellular, side of the membrane at concentrations as low as 10 nM. In contrast, DHS-I had no effect on several other types of potassium channels or membrane transporters. This natural product is the first example of a high-affinity activator of calcium-dependent potassium channels and is the most potent known potassium channel opener.

Animals↗
PubMed1993

Inhibition of beta-adrenoceptor agonist relaxation of airway smooth muscle by Ca(2+)-activated K+ channel blockers.

In isolated guinea pig trachea contracted by 0.5 mM acetylcholine, the cumulative relaxant concentration-response curves to the beta 2-adrenoceptor agonist, salbutamol, were shifted to the right by depolarizing concentrations of KCl, as well as by charybdotoxin, iberiotoxin and tetraethylammonium ion, which are antagonists of the high-conductance Ca(2+)-activated K+ channel. The shifts produced by KCl (40 mM), charybdotoxin (100 nM), iberiotoxin (50 nM), and tetraethylammonium ion (2 mM) were approximately 230-fold, 10-fold, 78-fold, and 8-fold, respectively. The blockade of beta 2-adrenoceptor agonist-induced relaxation by these agents was totally reversed by 0.3 microM nifedipine. Similar reversal was obtained with either 100 microM CdCl2, or low Ca2+ (50 microM) Krebs medium. These data suggest that charybdotoxin, iberiotoxin and tetraethylammonium ion, like KCl, cause membrane depolarization which in turn activates voltage-dependent Ca2+ channels. The influx of Ca2+ via these channels provides an additional mode to that of release of intracellular Ca2+ evoked by acetylcholine for maintaining cell Ca2+ concentration at a high level. This is apparently sufficient to account functionally for the blockade of beta 2-adrenoceptor agonist-induced relaxation. In view of this interpretation regarding the action of Ca(2+)-activated K+ channel antagonists, earlier proposals ascribing the relaxant effect of beta 2-adrenoceptor agonists strictly to activation of these channels must be reevaluated.

Acetylcholine↗
PubMed1993

Mobilization of intracellular calcium in cultured vascular smooth muscle cells by uridine triphosphate and the calcium ionophore A23187.

The known action of uridine triphosphate (UTP) to contract some types of vascular smooth muscle, and the present finding that it is more potent than adenosine triphosphate in eliciting an increase in cytosolic Ca2+ concentration in aortic smooth muscle, led us to investigate the mode of action of this nucleotide. With this aim, cultured bovine aorta cells were subjected to patch-clamp methodologies under various conditions. Nucleotide-induced variations in cytosolic Ca2+ were monitored by using single channel recordings of the high conductance Ca(2+)-activated K+ (Maxi-K) channel within on-cell patches as a reporter, and whole-cell currents were measured following perforation of the patch. In cells bathed in Na(+)-saline, UTP (> 30 nM) induced an inward current, and both Maxi-K channel activity and unitary current amplitude of the Maxi-K channel transiently increased. Repetitive exposures elicited similar responses when 5 to 10 min wash intervals were allowed between challenges of nucleotide. Oscillations in channel activity, but not oscillation in current amplitude were frequently observed with UTP levels > 0.1 microM. Cells bathed in K+ saline (150 mM) were less sensitive to UTP (approximately 5-fold), and did not show an increase in unitary Maxi-K current amplitude. Since the increase in amplitude occurs due to depolarization of the cell membrane, a change in amplitude was not observed in cells previously depolarized with K+ saline. The enhancement of Maxi-K channel activity in the presence of UTP was not diminished by Ca2+ entry blockers or by removal of extracellular Ca2+. However, in the latter case, repetitive responses progressively declined. These observations, as well as data comparing the action of low concentrations of Ca2+ ionophores (< 5 microM) to that of UTP indicate that both agents elevate cytosolic Ca2+ by mobilization of this ion from intracellular pools. However, the Ca2+ ionophore did not cause membrane depolarization, and thus did not change unitary current amplitude. The effect of UTP on Maxi-K channel activity and current amplitude was blocked by pertussis toxin and by phorbol 12-myristate 13-acetate (PMA), but was not modified by okadaic acid, or by inhibitors of protein kinase C (PKC). Our data support a model in which a pyrimidinergic receptor is coupled to a G protein, and this interaction mediates release of Ca2+ from intracellular pools, presumably via the phosphatidyl inositol pathway. This also results in activation of membrane channels that give rise to an inward current and depolarization. Ultimately, smooth muscle contraction ensues.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1992

Selective blockers of voltage-gated K+ channels depolarize human T lymphocytes: mechanism of the antiproliferative effect of charybdotoxin.

Charybdotoxin (ChTX), a K+ channel blocker, depolarizes human peripheral T lymphocytes and renders them insensitive to activation by mitogen. We observed four types of K+ channels in human T cells: one voltage-activated, and three Ca(2+)-activated. To discern the mechanism by which ChTX depolarizes T cells, we examined the sensitivity of both the voltage-activated and Ca(2+)-activated K+ channels to ChTX and other peptide channel blockers. All four types were blocked by ChTX, whereas noxiustoxin and margatoxin blocked only the voltage-activated channels. All three toxins, however, produced equivalent depolarization in human T cells. We conclude that the membrane potential of resting T cells is set by voltage-activated channels and that blockade of these channels is sufficient to depolarize resting human T cells and prevent activation.

Calcium↗
PubMed1990

Synthesis and structural characterization of charybdotoxin, a potent peptidyl inhibitor of the high conductance Ca2(+)-activated K+ channel.

Charybdotoxin (ChTX), a potent inhibitor of the high conductance Ca2(+)-activated K+ channel (PK,Ca) is a highly basic peptide isolated from venom of the scorpion Leiurus quinquestriatus hebraeus, whose primary structure has been determined (Gimenez-Gallego, G., Navia, M. A., Reuben, J. P., Katz, G. M., Kaczorowski, G. J., and Garcia, M. L. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 3329-3333). The synthesis of this peptide using continuous flow solid phase fluorenylmethyloxycarbonyl-pentafluorophenyl ester methodology has now been achieved. The 1-37-amino acid hexasulfhydryl peptide oxidizes readily to give the tricyclic disulfide structure in good yield. This folded synthetic material is identical to native toxin based on three criteria: co-migration with ChTX on reversed phase high performance liquid chromatography (HPLC); competitive inhibition of 125I-labeled monoiodotyrosine charybdotoxin binding to bovine aortic sarcolemmal membrane vesicles with a Ki (10 pM) identical to that of native toxin; blockade of PK,Ca activity in excised outside-out patches from bovine aortic smooth muscle with the potency and inhibitory properties characteristic of ChTX (i.e. appearance of silent periods interdispersed with normal bursts of channel activity in single channel recordings). Selective enzymatic digestion of native or synthetic ChTX by simultaneous exposure to chymotrypsin and trypsin yields identical reversed phase HPLC profiles. Analysis of the sequence and amino acid composition of the resulting fragments defines a disulfide bond arrangement (Cys7-Cys28, Cys13-Cys33, Cys17-Cys35) which differs from that previously suggested. This configuration predicts a highly folded tertiary structure for ChTX which, together with observations from electrophysiological and binding experiments, suggests a possible mechanism by which ChTX interacts with PK,Ca to block channel function.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of a unique, potent, peptidyl probe for the high conductance calcium-activated potassium channel from venom of the scorpion Buthus tamulus.

An inhibitor of the high conductance, Ca2(+)-activated K+ channel (PK,Ca) has been purified to homogeneity from venom of the scorpion Buthus tamulus by a combination of ion exchange and reversed-phase chromatography. This peptide, which has been named iberiotoxin (IbTX), is one of two minor components of the crude venom which blocks PK,Ca. IbTX consists of a single 4.3-kDa polypeptide chain, as determined by polyacrylamide gel electrophoresis, analysis of amino acid composition, and Edman degradation. Its complete amino acid sequence has been defined. IbTX displays 68% sequence homology with charybdotoxin (ChTX), another scorpion-derived peptidyl inhibitor of PK,Ca, and, like this latter toxin, its amino terminus contains a pyroglutamic acid residue. However, IbTX possesses 4 more acidic and 1 less basic amino acid residue than does ChTX, making this toxin much less positively charged than the other peptide. In single channel recordings, IbTX reversibly blocks PK,Ca in excised membrane patches from bovine aortic smooth muscle. It acts exclusively at the outer face of the channel and functions with an IC50 of about 250 pM. Block of channel activity appears distinct from that of ChTX since IbTX decreases both the probability of channel opening as well as the channel mean open time. IbTX is a selective inhibitor of PK,Ca; it does not block other types of voltage-dependent ion channels, especially other types of K+ channels that are sensitive to inhibition by ChTX. IbTX is a partial inhibitor of 125I-ChTX binding in bovine aortic sarcolemmal membrane vesicles (Ki = 250 pM). The maximal extent of inhibition that occurs is modulated by K+, decreasing as K+ concentration is raised, but K+ does not affect the absolute inhibitory potency of IbTX. A Scatchard analysis indicates that IbTX functions as a noncompetitive inhibitor of ChTX binding. Taken together, these data suggest that IbTX interacts at a distinct site on the channel and modulates ChTX binding by an allosteric mechanism. Therefore, IbTX defines a new class of peptidyl inhibitor of PK,Ca with unique properties that make it useful for investigating the characteristics of this channel in target tissues.

Amino Acid Sequence↗
PubMed1990

Ca2(+)-activated K+ channels in bovine aortic smooth muscle and GH3 cells: properties and regulation by guanine nucleotides.

Research directed towards discovering agents that enhance PKCa channel activity has followed two courses. One has been defining properties of the channel, as well as making use of toxins that are specific probes for characterizing channel binding sites and for isolating and purifying the channel. The second has been a study of the regulation of PKCa channels by guanine nucleotides. The following list summarizes the major findings and conclusions that have been drawn to date. (1) The high conductance (266 +/- 11.5 pS) PKCa channel of bovine aortic smooth muscle, which is the most abundant of the 3 kinds of PKCa channels observed, has properties very similar to high conductance PKCa channels found in other cell types. (2) Charybdotoxin (ChTX), a rather specific probe for PKCa channels, has been purified to homogeneity, characterized in terms of primary sequence, labeled with 125I, and used to study binding of ChTX to the receptor/channel complex. (3) A second high affinity toxin probe, with distinct biological activity, IbTX-I, has been purified to homogeneity, and its amino acid sequence determined. One of its distinct actions is to enhance channel activity at low nM levels, and to block the channel at concentrations above approximately 5 nM. (4) A number of vasodilators having the common action of increasing cytosolic cGMP levels, have been found to enhance PKCa channel activity in on-cell patches. (5) While all of the guanine nucleotides have been noted to enhance channel activity in excised patches at high concentrations (500 uM), only GMP is effective in the 10-100 uM range. (6) These data have led us to conclude that GMP is a physiological modulator of PKCa channels, and that it is most likely the second messenger for mediating the vasodilator-induced potentiation of channel activity.

Animals↗
PubMed1989

Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Evidence for a direct association with the high conductance calcium-activated potassium channel.

Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.

Animals↗
PubMed1988

Interaction of tetrandrine with slowly inactivating calcium channels. Characterization of calcium channel modulation by an alkaloid of Chinese medicinal herb origin.

Tetrandrine, a bis-benzylisoquinoline alkaloid derived from the Chinese medicinal herb Stephania tetrandra, is a putative Ca2+ entry blocker whose mechanism of action is unknown. To investigate this mechanism, the effects of tetrandrine were characterized on binding of three chemical classes of Ca2+ entry blockers in cardiac sarcolemmal membrane vesicles. In the range 25-37 degrees C, tetrandrine completely blocks diltiazem binding, partially inhibits D-600 binding, and markedly stimulates nitrendipine binding, with greatest enhancement occurring at 37 degrees C. The potency of tetrandrine is increased 10-fold as temperature is raised from 25 to 37 degrees C. Scatchard analyses indicate that inhibition of diltiazem binding and stimulation of nitrendipine binding result from changes in ligand affinities while inhibition of D-600 binding is due to both an increase in KD and decrease in Bmax of aralkylamine receptors. Ligand dissociation studies reveal that tetrandrine increases D-600 off-rates, decreases nitrendipine off-rates, but has no effect on diltiazem dissociation kinetics. In addition, tetrandrine reversibly blocks inward Ca2+ currents through L-type Ca2+ channels in GH3 anterior pituitary cells. These results indicate that tetrandrine interacts directly at the benzothiazepine-binding site of the Ca2+ entry blocker receptor complex and allosterically modulates ligand binding at other receptors in this complex. These findings suggest that tetrandrine is a structurally unique natural product Ca2+ entry blocker and provide a rationale explanation for the therapeutic effectiveness of this agent.

Alkaloids↗
PubMed1988

Purification, sequence, and model structure of charybdotoxin, a potent selective inhibitor of calcium-activated potassium channels.

Charybdotoxin (ChTX), a protein present in the venom of the scorpion Leiurus quinquestriatus var. hebraeus, has been purified to homogeneity by a combination of ion-exchange and reversed-phase chromatography. Polyacrylamide gel electrophoresis, amino acid analysis, and complete amino acid sequence determination of the pure protein reveal that it consists of a single polypeptide chain of 4.3 kDa. Purified ChTX is a potent and selective inhibitor of the approximately 220-pS Ca2+-activated K+ channel present in GH3 anterior pituitary cells and primary bovine aortic smooth muscle cells. The toxin reversibly blocks channel activity by interacting at the external pore of the channel protein with an apparent Kd of 2.1 nM. The primary structure of ChTX is similar to a number of neurotoxins of diverse origin, which suggests that ChTX is a member of a superfamily of proteins that modify ion-channel activities. On the basis of this similarity, the three-dimensional structure of ChTX has been modeled from the known crystal structure of alpha-bungarotoxin. These studies indicate that ChTX is useful as a probe of Ca2+-activated K+-channel function and suggest that the proposed tertiary structure of ChTX may provide insight into the mechanism of channel block.

Amino Acid Sequence↗
PubMed1988

Guanosine 5'-monophosphate modulates gating of high-conductance Ca2+-activated K+ channels in vascular smooth muscle cells.

Ca2+-activated K+ channels (PKCa channels) account for the predominant K+ permeability of many types of smooth muscle cells. When activated, they oppose depolarization due to Na+ and Ca2+ channel activity. Several vasodilatory agents that increase intracellular cGMP levels (e.g., nitroprusside, adenosine, and atrial natriuretic factor) enhance the activity of these high-conductance PKCa channels in on-cell patches of bovine aortic smooth muscle cells. In addition, dibutyryl-cGMP (1.0 mM) causes a similar increase in channel activity. To pursue the mechanism of channel modulation by these agents, a series of guanine and adenine nucleotides were evaluated by using inside-out excised patches. Whereas cAMP, AMP, ADP, and ATP were ineffective, all of the corresponding guanine nucleotides potentiated PKCa channel activity when tested at a high concentration (500 microM). However, only GMP consistently enhanced channel activity in the 1-100 microM range by increasing the percent open time and frequency of opening of these channels over a wide range of potentials and Ca2+ levels without affecting single-channel conductance. Thus, GMP is a potent modulator of PKCa channels and it, rather than cGMP, may mediate the action of the vasodilators examined in this study.

Animals↗
PubMed1987

Currents carried by sodium ions through transient calcium channels in clonal GH3 pituitary cells.

The transient or T-type Ca channel currents of voltage-clamped clonal (GH3) pituitary cells were studied after blockade of Na and K channels. Removal of Ca ions from the bathing medium abolished the Ca currents and allowed Na ions to convey current through the transient channels. These Na currents (I Na,t) were activated at threshold potentials of ca. -85 mV, reached peak amplitudes (up to 2,000 pA) at -60 mV, and were 50% inactivated at holding membrane potentials of -110 to -120 mV. The time constant for inactivation of the maximal I Na,t was 12 +/- 2.4 msec (compared with 22.1 =/- 0.85 msec for maximal I Ca,t). The I Na,t was insensitive to tetrodotoxin (1 micron) and to nimodipine (200 nM) but was abolished by 100 micron external [Ca2+]. It is suggested that high affinity Ca binding sites in the channel modulate the ion selectivity and the current flow through T-type Ca channels.

Action Potentials↗
PubMed1987

Effects of neomycin on calcium channel currents in clonal GH3 pituitary cells.

Membrane Ca currents were recorded from voltage-clamped clonal (GH3) pituitary cells under conditions where currents through Na and K channels were abolished. Two Ca currents, with distinct kinetics and voltage dependence for activation and inactivation, were identified. Neomycin, an aminoglycoside polycation, inhibited both the "transient" (ICa,t) and the "slowly-inactivating" (ICa,s) Ca currents in a dose-dependent manner (100-1,000 microM). The blockade was reversible and ICa,s was more sensitive to neomycin than ICa,t. The inhibition of ICa,s was frequency and time-independent, and was not affected by changes in the holding membrane potential (-35 to -100 mV). Neomycin did not affect the voltage dependence for inactivation of ICa,t. The blockade of both Ca currents by neomycin is ascribed to the general property of aminoglycosides to compete with, and displace Ca ions from membrane binding sites that determine the currents and selectivity of Ca channels. Because comparable concentrations of neomycin were required for blocking the currents conveyed by Ca or, in the absence of external divalent cations, by Na ions through the slowly-inactivating Ca channels, we suggest that the neomycin binding sites are distinct from the high-affinity transition sites within the Ca channel path.

Cells, Cultured↗
PubMed1986

Binding of Ca2+ entry blockers to cardiac sarcolemmal membrane vesicles. Characterization of diltiazem-binding sites and their interaction with dihydropyridine and aralkylamine receptors.

Stereospecific saturable and reversible binding of d-cis-diltiazem has been demonstrated in cardiac sarcolemmal membrane vesicles. Analysis of binding by either equilibrium or kinetic techniques indicates the presence of a single class of benzothiazepine receptors which bind diltiazem with a KD of 80 nM at 25 degrees C. Benzothiazepine receptors copurify with other sarcolemmal marker activities and exist in a complex with distinct receptors for dihydropyridine and aralkylamine Ca2+ entry blockers in a 1:1:1 stoichiometry. Ligand binding to one receptor of this complex influences binding reactions at the other two sites in a manner that depends on ambient temperature. Binding of either dihydropyridine agonists or antagonists causes partial inhibition of diltiazem binding at 25 degrees C (Bmax effect), while most dihydropyridine antagonists stimulate and agonists inhibit diltiazem binding at 37 degrees C (both are KD effects). This temperature-dependent change in receptor coupling was confirmed by Scatchard analyses and study of diltiazem dissociation kinetics. Verapamil, interacting at the aralkylamine receptor, inhibits diltiazem binding equivalently at 25 and 37 degrees C (KD effects). In addition, both classes of dihydropyridines inhibit verapamil binding in a temperature-independent fashion, as does diltiazem (all are KD effects). Allosteric coupling between benzothiazepine and dihydropyridine receptors is manifested in cardiac muscle since the negative inotropic potency of diltiazem is increased by nitrendipine and decreased by 4-(O-trifluromethy(phenyl)-2,6-dimethyl-5-nitro-1,4-dihydropyridin e-3- carboxylic acid, methyl ester. These results suggest a model in which the Ca2+ entry blocker receptor complex undergoes a change between 25 and 37 degrees C so that at the latter temperature all sites are directly coupled. Allosteric coupling may have important consequences in vivo since it can be detected in functional assays of Ca2+ channel activity.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Caffeine inhibition of calcium accumulation by the sarcoplasmic reticulum in mammalian skinned fibers.

Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca2+ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca2+ exceeds 0.3 to 0.4 microM, when free ATP exceeds 0.8 to 1 mM, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5 mM and is half-maximal at approximately 0.5 mM. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3 mM. Varying free Mg2+ (0.1 to 2 mM) or MgATP (0.03 to 10 mM) has no effect on inhibition. Average residual uptake rates in the presence of 5 mM caffeine at pCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca2+ concentration dependence of Ca uptake (K0.5, 0.5 to 0.8 microM; Hill n, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.

Adenosine Triphosphate↗