Upregulation of dorsal root ganglion (alpha)2(delta) calcium channel subunit and its correlation with allodynia in spinal nerve-injured rats.

Peripheral nerve injury can lead to a persistent neuropathic pain state in which innocuous tactile stimulation elicits pain behavior (tactile allodynia). Spinal administration of the anticonvulsant gabapentin suppresses allodynia by an unknown mechanism. In vitro studies indicate that gabapentin binds to the alpha(2)delta-1 (hereafter referred to as alpha(2)delta) subunit of voltage-gated calcium channels. We hypothesized that nerve injury may result in altered alpha(2)delta subunit expression in spinal cord and dorsal root ganglia (DRGs) and that this change may play a role in neuropathic pain processing. Using a rat neuropathic pain model in which gabapentin-sensitive tactile allodynia develops after tight ligation of the left fifth and sixth lumbar spinal nerves, we found a >17-fold, time-dependent increase in alpha(2)delta subunit expression in DRGs ipsilateral to the nerve injury. Marked alpha(2)delta subunit upregulation was also evident in rats with unilateral sciatic nerve crush, but not dorsal rhizotomy, indicating a peripheral origin of the expression regulation. The increased alpha(2)delta subunit expression preceded the allodynia onset and diminished in rats recovering from tactile allodynia. RNase protection experiments indicated that the DRG alpha(2)delta regulation was at the mRNA level. In contrast, calcium channel alpha(1B) and beta(3) subunit expression was not co-upregulated with the alpha(2)delta subunit after nerve injury. These data suggest that DRG alpha(2)delta regulation may play an unique role in neuroplasticity after peripheral nerve injury that may contribute to allodynia development.

Biophysical properties, pharmacology, and modulation of human, neuronal L-type (alpha(1D), Ca(V)1.3) voltage-dependent calcium currents.

Voltage-dependent calcium channels (VDCCs) are multimeric complexes composed of a pore-forming alpha(1) subunit together with several accessory subunits, including alpha(2)delta, beta, and, in some cases, gamma subunits. A family of VDCCs known as the L-type channels are formed specifically from alpha(1S) (skeletal muscle), alpha(1C) (in heart and brain), alpha(1D) (mainly in brain, heart, and endocrine tissue), and alpha(1F) (retina). Neuroendocrine L-type currents have a significant role in the control of neurosecretion and can be inhibited by GTP-binding (G-) proteins. However, the subunit composition of the VDCCs underlying these G-protein-regulated neuroendocrine L-type currents is unknown. To investigate the biophysical and pharmacological properties and role of G-protein modulation of alpha(1D) calcium channels, we have examined calcium channel currents formed by the human neuronal L-type alpha(1D) subunit, co-expressed with alpha(2)delta-1 and beta(3a), stably expressed in a human embryonic kidney (HEK) 293 cell line, using whole cell and perforated patch-clamp techniques. The alpha(1D)-expressing cell line exhibited L-type currents with typical characteristics. The currents were high-voltage activated (peak at +20 mV in 20 mM Ba2+) and showed little inactivation in external Ba2+, while displaying rapid inactivation kinetics in external Ca2+. The L-type currents were inhibited by the 1,4 dihydropyridine (DHP) antagonists nifedipine and nicardipine and were enhanced by the DHP agonist BayK S-(-)8644. However, alpha(1D) L-type currents were not modulated by activation of a number of G-protein pathways. Activation of endogenous somatostatin receptor subtype 2 (sst2) by somatostatin-14 or activation of transiently transfected rat D2 dopamine receptors (rD2(long)) by quinpirole had no effect. Direct activation of G-proteins by the nonhydrolyzable GTP analogue, guanosine 5'-0-(3-thiotriphospate) also had no effect on the alpha(1D) currents. In contrast, in the same system, N-type currents, formed from transiently transfected alpha(1B)/alpha(2)delta-1/beta(3), showed strong G-protein-mediated inhibition. Furthermore, the I-II loop from the alpha(1D) clone, expressed as a glutathione-S-transferase (GST) fusion protein, did not bind Gbetagamma, unlike the alpha(1B) I-II loop fusion protein. These data show that the biophysical and pharmacological properties of recombinant human alpha(1D) L-type currents are similar to alpha(1C) currents, and these currents are also resistant to modulation by G(i/o)-linked G-protein-coupled receptors.

Characterization of the recombinant human neuronal nicotinic acetylcholine receptors alpha3beta2 and alpha4beta2 stably expressed in HEK293 cells.

HEK293 cells were stably transfected with the cDNAs encoding full-length human neuronal nicotinic acetylcholine receptor (nAChR) subunit combinations alpha3beta2 or alpha4beta2. [(3)H]-(+/-)Epibatidine ([(3)H]-(+/-)EPI) bound to membranes from A3B2 (alpha3beta2) and A4B2.2 (alpha4beta2) cells with K(d) values of 7.5 and 33.4 pM and B(max) values of 497 and 1564 fmol/mg protein, respectively. Concentration-dependent increases in intracellular free Ca(2+) concentration were elicited by nAChR agonists with a rank order of potency of EPI>1,1-dimethyl-4-phenylpiperazinium (DMPP)>nicotine (NIC)=suberyldicholine (SUB)>cytisine (CYT)=acetylcholine (ACh) for A3B2 cells and EPI>CYT=SUB=NIC=DMPP>ACh for A4B2.2 cells. Antagonists of nAChRs blocked NIC-induced responses with a rank order of potency of d-tubocurarine (d-Tubo)=mecamylamine (MEC)>dihydro-beta-erythroidine (DHbetaE) in A3B2 cells and MEC=DHbetaE>d-Tubo in A4B2.2 cells. Whole-cell patch clamp recordings indicate that the decay rate of macroscopic ACh-induced currents is faster in A3B2 than in A4B2.2 cells and that A3B2 cells are less sensitive to ACh than A4B2.2 cells. ACh currents elicited in alpha3beta2 and alpha4beta2 human nAChRs are maximally potentiated at 20 and 2 mM external Ca(2+), respectively. Our results indicate that stably expressed alpha3beta2 and alpha4beta2 human nAChRs are pharmacologically and functionally distinct.

Functional consequences of mutations in the human alpha1A calcium channel subunit linked to familial hemiplegic migraine.

Mutations in alpha1A, the pore-forming subunit of P/Q-type calcium channels, are linked to several human diseases, including familial hemiplegic migraine (FHM). We introduced the four missense mutations linked to FHM into human alpha1A-2 subunits and investigated their functional consequences after expression in human embryonic kidney 293 cells. By combining single-channel and whole-cell patch-clamp recordings, we show that all four mutations affect both the biophysical properties and the density of functional channels. Mutation R192Q in the S4 segment of domain I increased the density of functional P/Q-type channels and their open probability. Mutation T666M in the pore loop of domain II decreased both the density of functional channels and their unitary conductance (from 20 to 11 pS). Mutations V714A and I1815L in the S6 segments of domains II and IV shifted the voltage range of activation toward more negative voltages, increased both the open probability and the rate of recovery from inactivation, and decreased the density of functional channels. Mutation V714A decreased the single-channel conductance to 16 pS. Strikingly, the reduction in single-channel conductance induced by mutations T666M and V714A was not observed in some patches or periods of activity, suggesting that the abnormal channel may switch on and off, perhaps depending on some unknown factor. Our data show that the FHM mutations can lead to both gain- and loss-of-function of human P/Q-type calcium channels.

Structure and functional characterization of a novel human low-voltage activated calcium channel.

We have isolated and characterized overlapping cDNAs encoding a novel, voltage-gated Ca2+ channel alpha1 subunit, alpha1H, from a human medullary thyroid carcinoma cell line. The alpha1H subunit is structurally similar to previously described alpha1 subunits. Northern blot analysis indicates that alpha1H mRNA is expressed throughout the brain, primarily in the amygdala, caudate nucleus, and putamen, as well as in several nonneuronal tissues, with relatively high levels in the liver, kidney, and heart. Ba2+ currents recorded from human embryonic kidney 293 cells transiently expressing alpha1H activated at relatively hyperpolarized potentials (-50 mV), rapidly inactivated (tau = 17 ms), and slowly deactivated. Similar results were observed in Xenopus oocytes expressing alpha1H. Single-channel measurements in human embryonic kidney 293 cells revealed a single-channel conductance of approximately 9 pS. These channels are blocked by Ni2+ (IC50 = 6.6 microM) and the T-type channel antagonists mibefradil (approximately 50% block at 1 microM) and amiloride (IC50 = 167 microM). Thus, alpha1H-containing channels exhibit biophysical and pharmacological properties characteristic of low voltage-activated, or T-type, Ca2+ channels.

Characterization of human recombinant neuronal nicotinic acetylcholine receptor subunit combinations alpha2beta4, alpha3beta4 and alpha4beta4 stably expressed in HEK293 cells.

Human embryonic kidney (HEK293) cells were transfected with cDNA encoding the human beta4 neuronal nicotinic acetylcholine (ACh) receptor subunit in pairwise combination with human alpha2, alpha3 or alpha4 subunits. Cell lines A2B4, A3B4.2 and A4B4 were identified that stably express mRNA and protein corresponding to alpha2 and beta4, to alpha3 and beta4 and to alpha4 and beta4 subunits, respectively. Specific binding of [3H]epibatidine was detected in A2B4, A3B4.2 and A4B4 cells with Kd (mean +/- S.D. in pM) values of 42 +/- 10, 230 +/- 12 and 187 +/- 29 and with Bmax (fmol/mg protein) values of 1104 +/- 338, 2010 +/- 184 and 3683 +/- 1450, respectively. Whole-cell patch-clamp recordings in each cell line demonstrated that (-)nicotine (Nic), ACh, cytisine (Cyt) and 1, 1-dimethyl-4-phenylpiperazinium iodide (DMPP) elicit transient inward currents. The current-voltage (I-V) relation of these currents showed strong inward rectification. Pharmacological characterization of agonist-induced elevations of intracellular free Ca++ concentration revealed a distinct rank order of agonist potency for each subunit combination as follows: alpha2beta4, (+)epibatidine (Epi) > Cyt > suberyldicholine (Sub) = Nic = DMPP; alpha3beta4, Epi > DMPP = Cyt = Nic = Sub; alpha4beta4, Epi > Cyt = Sub > Nic > DMPP. The noncompetitive antagonists mecamylamine and d-tubocurarine did not display subtype selectivity. In contrast, the Kb value for the competitive antagonist dihydro-beta-erythroidine (DHbetaE) was highest at alpha3beta4 compared with alpha2beta4 or alpha4beta4 receptors. These data illustrate that the A2B4, A3B4.2 and A4B4 stable cell lines are powerful tools for examining the functional and pharmacological properties of human alpha2beta4, alpha3beta4 and alpha4beta4 neuronal nicotinic receptors.

Rat group I metabotropic glutamate receptors inhibit neuronal Ca2+ channels via multiple signal transduction pathways in HEK 293 cells.

We have shown previously that metabotropic glutamate receptors with group I-like pharmacology couple to N-type and P/Q-type calcium channels in acutely isolated cortical neurons using G proteins most likely belonging to the Gi/Go subclass. To better understand the potential mechanisms forming the basis for group I mGluR modulation of voltage-gated calcium channels in the CNS, we have examined the ability of specific mGluRs to couple to neuronal N-type (alpha1B-1/alpha2delta/beta1b) and P/Q-type (alpha1A-2/alpha2delta/beta1b) voltage-gated calcium channels in an HEK 293 heterologous expression system. Using the whole cell patch-clamp technique where intracellular calcium is buffered to low levels, we have shown that group I receptors inhibit both N-type and P/Q-type calcium channels in a voltage-dependent fashion. Similar to our observations in cortical neurons, this voltage-dependent inhibition is mediated almost entirely by N-ethylmaleimide (NEM)-sensitive heterotrimeric G proteins, strongly suggesting that these receptors can use Gi/Go-like G proteins to couple to N-type and P/Q-type calcium channels. However, inconsistent with the apparent NEM sensitivity of group I modulation of calcium channels, modulation of N-type channels in group I mGluR-expressing cells was only partially sensitive to pertussis toxin (PTX), indicating the potential involvement of both PTX-sensitive and -resistant G proteins. The PTX-resistant modulation was voltage dependent and entirely resistant to NEM and cholera toxin. A time course of treatment with PTX revealed that this toxin caused group I receptors to slowly shift from using a primarily NEM-sensitive G protein to using a NEM-resistant form. The PTX-induced switch from NEM-sensitive to -resistant modulation was also dependent on protein synthesis, indicating some reliance on active cellular processes. In addition to these voltage-dependent pathways, perforated patch recordings on group I mGluR-expressing cells indicate that another slowly developing, calcium-dependent form of modulation for N-type channels may be seen when intracellular calcium is not highly buffered. We conclude that group I mGluRs can modulate neuronal Ca2+ channels using a variety of signal transduction pathways and propose that the relative contributions of different pathways may exemplify the diversity of responses mediated by these receptors in the CNS.

Pharmacological characterization of SIB-1765F: a novel cholinergic ion channel agonist.

Nicotine, the prototypical agonist for neuronal nicotinic acetylcholine receptors (NAChR), nonselectively activates NAChR limiting its use in elucidating the function of NAChR subtypes. SIB-1765F is a subtype selective NAChR agonist that displaces [3H]-nicotine binding with an IC50 of 4.6 nM and [3H]-cytisine binding with an IC50 of 12.2 nM which is 2000- to 6000-fold lower than its displacement of [3H]-QNB or [125I]-alpha-bungarotoxin. SIB-1765F did not inhibit human or rat cholinesterases or the uptake of [3H]-DA in synaptosomal preparations. SIB-1765F mimicked (-)-nicotine in stimulating [3H]-DA release from rat striatal and olfactory tubercle slices, with EC50 values of 99.6 and 39.6 microM, respectively. Such stimulation was sensitive to mecamylamine and DH beta E. SIB-1765F also released endogenous DA in the striatum and the nucleus accumbens as measured by in vivo microdialysis. SIB-1765F was less efficacious than (-)-nicotine at stimulating [3H]-NE release from rat hippocampal slices; in contrast, SIB-1765F increased [3H]-NE release from rat thalamic and cortical slices with efficacies approaching those of (-)-nicotine. Similar to (-)-nicotine and (+/-)-epibatidine, subcutaneous administration of SIB-1765F increased the turnover rate of dopamine ex vivo both in the striatum and olfactory tubercles in a mecamylamine-sensitive manner. Because the release of striatal DA and hippocampal NE appears to be regulated by distinct NAChR, differential effects of SIB-1765F on striatal DA and hippocampal NE release supports the NAChR subtype selectivity of SIB-1765F compared to (-)-nicotine. This is further demonstrated by observations showing that SIB-1765F has a higher affinity for h alpha 4 beta 2 NAChR relative to h alpha 4 beta 4 NAChRs in displacing [3H]-epibatidine binding and increasing cytosolic CA+2 concentration in cell lines stably expressing h alpha 4 beta 2 or h alpha 4 beta 4.

Relative contributions of G protein, channel, and receptor to voltage-dependent inhibition of neuronal N-type and P/Q-type calcium channels in HEK 293 cell lines.

The voltage-dependent modulation of neuronal voltage-gated calcium channels by heterotrimeric G protein-coupled receptors potentially provides a means for activity-dependent modulation of synaptic efficacy. Recent attention has focused upon the molecular mechanisms by which such G proteins influence the biophysical properties of calcium channels. We have used an HEK 293-based heterologous system which stably expresses human neuronal calcium channels to address the relative contributions of receptor, G protein, and channel to voltage-dependent inhibition. We find that the receptor and channel subtype only insignificantly influence the time it takes to re-establish modulation following voltage-dependent relief of inhibition. In contrast, the G protein subtype mediating inhibition appears to play a significant part in this process. These results emphasize the importance of G protein subtype in the modulation of neuronal calcium channels.

Histamine-induced Ca2+ entry precedes Ca2+ mobilization in bovine adrenal chromaffin cells.

The relationship between histamine-induced Ca2+ mobilization and Ca2+ entry in bovine adrenal chromaffin cells has been investigated. Stopped-flow fluorimetry of fura-2-loaded chromaffin cell populations revealed that 10 microM histamine promoted entry of Ca2+ or Mn2+ without measurable delay (< or = 20 ms), through a pathway that was insensitive to the dihydropyridine antagonist nifedipine. In the absence of extracellular Ca2+, or in the presence of 100 microM La3+, a blocker of receptor-mediated Ca2+ entry, 10 microM histamine triggered an elevation in intracellular calcium concentration ([Ca2+]i), but only after a delay of approx. 200 ms, which presumably represented the time required to mobilize intracellular Ca2+. These data suggested that histamine-induced bivalent-cation entry precedes extensive Ca2+ mobilization in chromaffin cells. In order to confirm that histamine can promote Ca2+ entry largely independently of mobilizing intracellular Ca2+, the ability of histamine to promote Ca2+ entry into cells whose intracellular Ca2+ store had been largely depleted was assessed. Fura-2-loaded chromaffin cells were treated with 10 microM ryanodine together with 40 mM caffeine, to deplete the hormone-sensitive Ca2+ store. This resulted in an approx. 95% inhibition of histamine-induced Ca2+ release. Under these conditions, histamine was still able to promote an entry of Ca2+ that was essentially indistinguishable from that promoted in control cells. In single cells, introduction of heparin (100 mg/ml), but not de-N-sulphated heparin (100 mg/ml), abolished the histamine-induced rise in [Ca2+]i. All these data suggest that histamine can induce G-protein- or inositol phosphate-dependent rapid (< or = 20 ms) Ca2+ entry without an extensive intracellular mobilization response in chromaffin cells, which points to activation of an entry mechanism distinct from the Ca(2+)-release-activated Ca2+ channel found in non-excitable cells.

Quantal Ca2+ mobilization by ryanodine receptors is due to all-or-none release from functionally discrete intracellular stores.

Low caffeine concentrations were unable to completely release the caffeine- and ryanodine-sensitive intracellular Ca2+ pool in intact adrenal chromaffin cells. This 'quantal' Ca2+ release is the same as that previously observed with inositol Ins(1,4,5)P3-induced Ca2+ release. The molecular mechanism underlying quantal Ca2+ release from the ryanodine receptor was investigated using fura-2 imaging of single chromaffin cells. Our data indicate that the intracellular caffeine-sensitive Ca2+ pool is composed of functionally discrete stores, that possess heterogeneous sensitivities to caffeine. These stores are mobilized by caffeine in a concentration-dependent fashion, and, when stimulated, individual stores release their Ca2+ in an 'all-or-none' manner. Such quantal Ca2+ release may be responsible for graded Ca2+ responses in single cells.

Quantal Ca2+ release from caffeine-sensitive stores in adrenal chromaffin cells.

In populations of fura-2-loaded chromaffin cells, caffeine caused a concentration-dependent increase in the intracellular Ca2+ concentration ([Ca2+]i), in the presence or absence of external Ca2+ ([Ca2+]o), that was saturable, reversible, and inhibited in a use-dependent fashion by ryanodine. These data confirm that caffeine mobilizes Ca2+ from the ryanodine-sensitive intracellular stores in chromaffin cells. In nominally Ca(2+)-free medium, sustained stimulation of cell populations or single cells with low caffeine concentrations failed to completely empty the caffeine-sensitive stores. In each case, there was a transient [Ca2+]i elevation, but a subsequent challenge with a higher caffeine concentration evoked a further [Ca2+]i rise, indicating that Ca2+ stores within individual cells were heterogeneous in their sensitivities to caffeine and that caffeine-induced Ca2+ release was quantal. The heterogeneous sensitivity was also demonstrated using ryanodine; pretreatment of cell populations with increasing caffeine concentrations with a constant ryanodine concentration, caused a dose-dependent irreversible inhibition of the response to the subsequent addition of a maximal caffeine concentration. We conclude that, within single chromaffin cells, intracellular Ca2+ stores are heterogeneous in their sensitivity to caffeine and the fraction of Ca2+ stores mobilized by caffeine increases in direct proportion to the caffeine concentration.

Fluoxetine-induced inhibition of synaptosomal [3H]5-HT release: possible Ca(2+)-channel inhibition.

Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K(+)-induced [3H]5-HT release from rat spinal cord and cortical synaptosomes at concentrations greater than 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K+ used to depolarize the synaptosomes and the concentration of external Ca2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [3H]5-HT release induced by the Ca(2+)-ionophore A 23187 or Ca(2+)-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K(+)-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca2+ channels and Ca2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [3H]nitrendipine to the dihydropyridine-sensitive L-type Ca2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K(+)-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K(+)-induced increase in intracellular free Ca2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K(+)-induced [3H]5-HT release by antagonizing voltage-dependent Ca2+ entry into nerve terminals.

The inositol 1,4,5-trisphosphate-forming agonist histamine activates a ryanodine-sensitive Ca2+ release mechanism in bovine adrenal chromaffin cells.

The role of a Ca(2+)-induced Ca2+ release (CICR) mechanism in the generation of agonist-induced increases of intracellular free Ca2+ concentration ([Ca2+]i) was studied in bovine adrenal chromaffin cells. In single cells, repetitive stimulations with caffeine at 200-s intervals evoked reproducible spikes of [Ca2+]i. Ryanodine, an agent that interacts with the CICR channel of muscle, inhibited the caffeine-induced spikes of [Ca2+]i in a "use-dependent" way. High affinity binding sites for [3H]ryanodine (Kd 3.3 nM, Bmax 26 fmol/mg protein) were also detected in membranes from chromaffin cells, supporting the presence of a caffeine- and ryanodine-sensitive CICR channel. Pretreatment of single cells with caffeine + ryanodine to reduce the size of the caffeine-sensitive Ca2+ compartment inhibited a subsequent spike of [Ca2+]i evoked by histamine, a D-myo-inositol 1,4,5-trisphosphate-forming agonist. This demonstrates that a significant portion of the Ca2+ released by histamine comes from a caffeine- and ryanodine-sensitive pool. Ryanodine inhibited by 50% the size of [Ca2+]i spikes evoked by repetitive stimulation with histamine and did so in a use-dependent manner. These data suggest that, in addition to D-myoinositol 1,4,5-trisphosphate, activation of a caffeine- and ryanodine-sensitive CICR channel participates in the generation of histamine-induced release of intracellular Ca2+.

The role of caffeine-sensitive Ca2+ stores in agonist- and inositol 1,4,5-trisphosphate-induced Ca2+ release from bovine adrenal chromaffin cells.

In single bovine adrenal chromaffin cells loaded with fura-2, histamine, angiotensin II (AII) and caffeine elicited large transient increases of intracellular free Ca2+ concentration [( Ca2+]i) in the absence of external Ca2+, with peak amplitudes averaging 726 +/- 138 (n = 14), 710 +/- 102 (n = 21) and 830 +/- 100 nM (n = 30) respectively. A substantial portion of the agonist-induced rise in [Ca2+]i depended on Ca2+ release from caffeine-sensitive stores, as pretreatment with caffeine diminished subsequent agonist responses by 90-95%. Conversely, pretreatment with histamine or AII decreased subsequent caffeine responses by 100% and 90% respectively. The effects of caffeine most likely resulted from activation of a Ca(2+)-induced Ca(2+)-release (CICR) process, whereas histamine and AII initially acted through generation of Ins(1,4,5)P3. The relationship of Ins(1,4,5)P3- and caffeine-sensitive Ca2+ pools was studied by using alpha-toxin-permeabilized chromaffin cells. Evidence was found for three non-mitochondrial, ATP-dependent, Ca2+ pools: one exclusively sensitive to Ins(1,4,5)P3 (pool 1), a second sensitive to both Ins(1,4,5)P3 and caffeine (pool 2), and a third exclusively sensitive to caffeine (pool 3). The existence of pools 1 and 3, and the ability of agonists such as histamine to discharge pool 3 completely, supports a two-pool model in which a caffeine-sensitive CICR mechanism plays a major role in the generation of agonist-induced Ca2+ spikes in bovine chromaffin cells.

Stilbene disulfonic acids. CD4 antagonists that block human immunodeficiency virus type-1 growth at multiple stages of the virus life cycle.

The stilbene disulfonic acids 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid and, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid bound the variable-1 immunoglobulin-like domain of CD4 on JM cells. The interaction blocked the binding of the anti-CD4 monoclonal antibody OKT4A and the envelope glycoprotein gp120 of the human immunodeficiency virus type-1 (HIV-1). DIDS inhibited the acute infection of CD4+ cells by HIV-1 with a potency (IC50 approximately 30 microM) similar to that which blocked gp120 binding (IC50 approximately 20 microM) to the cellular antigen. Pretreating uninfected CD4+ C8166 cells with DIDS blocked their fusion with chronically infected gp120+ cells. DIDS covalently and selectively modified lysine 90 of soluble CD4 and abolished the gp120-binding and antiviral properties of the recombinant protein. When added to cells productively infected with HIV-1, DIDS blocked virus growth and cleared cultures of syncytia without inhibiting cellular proliferation. The stilbene disulfonic acids are a novel class of site-specific CD4 antagonists that block multiple CD4-dependent events associated with acute and established HIV-1 infections.

Agonist-dependent patterns of cytosolic Ca2+ changes in single bovine adrenal chromaffin cells: relationship to catecholamine release.

The patterns of agonist-induced elevations of cytosolic free Ca2+ ([Ca2+]i) were characterized and compared by the use of single adrenal chromaffin cells. Initial histamine- or angiotensin II (AII)-induced elevations of [Ca2+]i were equal in magnitude (peaks 329 +/- 20 [SE] and 338 +/- 46 nM, respectively). These initial increases of [Ca2+]i were transient, insensitive to either Gd3+ or removing external Ca2+, and were primarily the result of Ca2+ release from intracellular stores. After the initial peak(s) of [Ca2+]i, a second phase of moderately elevated [Ca2+]i was observed, and this response was sensitive to either Gd3+ or removing external Ca2+, supporting a role for Ca2+ entry. In most cases, the second phase of elevated [Ca2+]i was sustained during histamine stimulation but transient during AII stimulation. Maintenance of the second phase was a property of the agonist rather than of the particular cell being stimulated. Thus, individual cells exposed sequentially to histamine and AII displayed distinct patterns of [Ca2+]i changes to each agonist, regardless of the order of addition. Histamine also stimulated twice as much [3H]catecholamine release as AII, and release was completely dependent on external Ca2+. Therefore, the ability of histamine and AII to sustain (or promote) Ca2+ entry appears to underlie their efficacy as secretagogues. These data provide evidence linking agonist-dependent patterns of [Ca2+]i changes in single cells with agonist-dependent functional responses.