A novel role for membrane potential in the modulation of intracellular Ca2+ oscillations in rat megakaryocytes.

1. The effect of membrane potential (Vm) on ADP-evoked [Ca2+]i oscillations was investigated in rat megakaryocytes, a non-excitable cell type recently shown to exhibit depolarisation-evoked Ca2+ release from intracellular stores during metabotropic purinoceptor stimulation. 2. Hyperpolarising voltage steps caused a transient fall in [Ca2+]i and either abolished Ca2+ oscillations or reduced the oscillation amplitude. These effects were observed in both the presence and absence of extracellular Ca2+ and also in Na+-free saline solutions, suggesting that hyperpolarisation leads to a reduction in the level of ADP-dependent Ca2+ release without a requirement for altered transmembrane Ca2+ fluxes. 3. In the presence of Ca2+ oscillations, depolarising voltage steps transiently enhanced the amplitude of Ca2+ oscillations. Following run-down of Ca2+ oscillations, depolarisation briefly restimulated oscillations. 4. Simultaneous [Ca2+]i and current-clamp recordings showed that Ca2+ and Vm oscillate in synchrony, with an average fluctuation of approximately 30-40 mV, due to activation and inactivation of Ca2+-dependent K+ channels. Application of a physiological oscillating Vm waveform to non-oscillating cells under voltage clamp stimulated [Ca2+]i oscillations. 5. Analysis of the relationship between [Ca2+]i and Vm showed a threshold for activation of hyperpolarisation at about 250-300 nM. The implications of this threshold in the interaction between Vm and Ca2+ release during oscillations are discussed. 6. We conclude that the ability of voltage to control release of endosomal Ca2+ in ADP-stimulated megakaryocytes is bipolar in nature. Our data suggest that Vm changes are active components of the feedback/feedforward mechanisms contributing to the generation of Ca2+ oscillations.

Depolarization-evoked Ca2+ release in a non-excitable cell, the rat megakaryocyte.

1. The effect of membrane potential on [Ca2+]i in rat megakaryocytes was studied using simultaneous whole-cell patch clamp and fura-2 fluorescence recordings. 2. Depolarization from -75 to 0 mV had no effect on [Ca2+]i in unstimulated cells, but evoked one or more spikes of Ca2+ increase (peak increase: 714 +/- 95 nM) during activation of metabotropic purinoceptors by 1 microM ADP. 3. The depolarization-evoked Ca2+ increase was present in Ca2+-free medium and also following removal of Na+. Thus depolarization mobilizes Ca2+ from an intracellular store without a requirement for altered Na+-Ca2+ exchange activity. 4. Intracellular dialysis with heparin blocked the depolarization-evoked Ca2+ increase, indicating a role for functional IP3 receptors. 5. Under current clamp, ADP caused the membrane potential to fluctuate between -43 +/- 1 and -76 +/- 1 mV. Under voltage clamp, depolarization from -75 to -45 mV evoked a transient [Ca2+]i increase (398 +/- 91 nM) during exposure to ADP. 6. We conclude that during stimulation of metabotropic purinoceptors, membrane depolarization over the physiological range can stimulate Ca2+ release from intracellular stores in the rat megakaryocyte, a non-excitable cell type. This may represent an important mechanism by which electrogenic influences can control patterns of [Ca2+]i increase.

Reversible and irreversible intracellular Ca2+ spiking in single isolated human platelets.

1. We have developed conditions that permit long duration recordings of [Ca2+]i in single, isolated human platelets and studied the reversibility of Ca2+i spiking following activation by physiological and artificial stimuli. 2. Fura-2-loaded platelets were immobilized at the tip of a saline-filled glass pipette using gentle suction. 'Contact' activation of Ca2+i spiking was observed in a proportion (11 %) of platelets, which continued for the duration of each recording (range 8-45 min). 3. Platelets that displayed constant, resting Ca2+i levels were used to test the effects of agonists. ADP (10 microM) increased [Ca2+]i in the form of either one to two spikes followed by an elevated plateau level (60 % of cells) or multiple Ca2+ spikes of irregular amplitude (40 % of cells). ADP-induced Ca2+i mobilization was completely reversible and repeatable. 4. Thrombin (1 u ml-1) evoked Ca2+i spiking in the majority (88 %) of platelets tested, which was not inhibited by perfusion of agonist-free saline throughout the recording period (range 8-67 min). 5. The clear difference in the reversibility of activation by different stimuli may reflect the distinct roles of individual agonists in haemostasis and have important consequences in the design of treatments for thrombosis.

ADP and inositol trisphosphate evoke oscillations of a monovalent cation conductance in rat megakaryocytes.

1. A combination of conventional whole-cell patch clamp recordings and fura-2 fluorescence photometry was used to study the membrane currents during oscillations of intracellular Ca2+ concentration ([Ca2+]i) in single rat megakaryocytes. 2. At a holding potential of -60 mV, in NaCl external saline and KCl internal saline with low levels of Ca2+ buffering, 10 microM ADP evoked [Ca2+]i oscillations and simultaneous Ca2+-gated K+ currents at a frequency of 3-10 spikes min-1. A smaller inward current was also activated, with a time course that identified this component as the inositol 1,4, 5-trisphosphate (IP3)-activated monovalent cation current previously demonstrated in rat megakaryocytes. 3. Cs+ replacement of internal K+ combined with 100 nM external charybdotoxin (CTX) abolished the outward currents and revealed that an inward current was also transiently activated during each [Ca2+]i spike. This underlying conductance was permeable to Na+ and Cs+, but possessed little or no permeability to Cl- or divalent cations. 4. Intracellular dialysis with IP3 (5-50 microM) activated the monovalent cationic conductance prior to release of Ca2+ from intracellular stores. The [Ca2+]i increase was associated with a second phase of cationic current, implying that both IP3 and Ca2+ can activate this conductance. Buffering of [Ca2+]i with BAPTA abolished the second phase of current, leaving monophasic spikes of inward current, often occurring at regular intervals. 5. These data demonstrate that a monovalent cation current, which results in Na+ influx under normal ionic conditions, oscillates in response to ADP receptor stimulation due to activation by both IP3 and [Ca2+]i. This provides a route for long-term Na+ entry in the megakaryocyte following stimulation of receptors coupled to phospholipase C activation and may play a role in cell shape change.

Comparison of the interaction of agmatine and crude methanolic extracts of bovine lung and brain with alpha 2-adrenoceptor binding sites.

1. In the present study we have evaluated whether alpha 2-adrenoceptor binding sites on bovine cerebral cortex membranes labelled by [3H]-clonidine, [3H]-idazoxan and [3H]-RX-821002 can distinguish between known agonists and antagonists. This model has then been used to compare the binding profiles of the putative non-catecholamine, clonidine-displacing substance (CDS), agmatine and crude methanolic extracts of bovine lung and brain. 2. Saturation studies carried out in the presence and absence of noradrenaline, 10 mumol 1(-1), revealed that the maximum number of binding sites on bovine cerebral cortex membranes for [3H]-idazoxan and [3H]-RX-821002 were approximately 60-80% greater than those for [3H]-clonidine (62.6 fmol mg-1 protein). Rauwolscine, the selective alpha 2-adrenoceptor antagonist, was approximately 100 fold more potent against each of the ligands than the selective alpha 1-adrenoceptor diastereoisomer, corynanthine. Also, the pKi value for the selective alpha 1-adrenoceptor prazosin against each ligand was less than 6. 3. Adrenaline, UK-14034, rauwolscine, corynanthine, RX-811059 and prazosin produced concentration-dependent inhibition of binding of all three 3H-ligands. The agonists, adrenaline and UK-14304, were approximately 5 and 10 fold less potent against [3H]-idazoxan and [3H]-RX-821002, respectively, than against [3H]-clonidine. In marked contrast, the antagonists, rauwolscine, corynanthine, RX-811059 and prazosin exhibited a different profile, being approximately 2-3 fold more potent against sites labelled by [3H]-RX-821002 and [3H]-idazoxan compared to sites labelled by [3H]-clonidine. 4. Agmatine and histamine produced a concentration-dependent displacement of [3H]-clonidine, [3H]-idazoxan and [3H]-RX-821002 binding to bovine cerebral cortex membranes. The pKi values for agmatine and histamine were independent of the 3H-ligand employed, approximately 4.8 and 4.5,respectively.5. Crude methanolic extracts of bovine brain and lung produced a concentration-dependent inhibition of [3H]-clonidine binding to bovine cerebral cortex membranes (>90%). Based on the volume of the extract that caused 50% inhibition of [3H]-clonidine binding, bovine lung contains 3 fold more CDS than bovine brain. Both extracts were at least 5 fold more potent against a2-adrenoceptor sites labelled by[3H]-clonidine than those labelled by [3H]-idazoxan and [3H]-RX-821002.6. All three 3H-ligands label the same population of alpha2-adrenoceptor binding sites on bovine cerebral cortex membranes, but [3H]-clonidine appears to label selectively the 'agonist' state of the sites: for which known agonists, adrenaline and UK-14304, exhibit a higher affinity. Our results indicate that neither agmatine nor histamine can account for the CDS activity present in crude extracts of bovine brain and lung. Moreover, these extracts appear to possess a binding profile similar to that of adrenaline and UK-14304, suggesting that they may possess agonist activity.

Evidence for the presence of a non-catecholamine, clonidine-displacing substance in crude, methanolic extracts of bovine brain and lung.

In the present study we have prepared crude, methanolic extracts of bovine lung and bovine brain and, using radioligand binding assays in conjunction with a number of simple chromatographic techniques, provided evidence for the presence of a non-catecholamine 'clonidine-displacing substance' (CDS). The level of CDS in lung extracts (9 units/g wet weight n = 11) is approximately 3 times that in the brain extracts. Furthermore, the effect of the crude, methanolic extracts are selective for non-adrenoceptor, imidazoline (labelled by [3H]-idazoxan) and alpha 2-adrenoceptor binding sites (labelled by [3H]-clonidine); both extracts are 5-10-fold more potent displacers of ligand binding to alpha 2-adrenoceptors compared with binding to opiate receptors (labelled by [3H]-etorphine) and practically inactive against alpha 1-adrenoceptor and muscarinic binding sites (labelled by [3H]-prazosin and [3H]-quinuclidinyl benzilate, respectively). With the exception of the non-adrenoceptor, imidazoline binding assay, which used rat kidney membranes labelled by [3H]-idazoxan in the presence of the alpha 2-adrenoceptor antagonist RS-15385-197, all radioreceptor assays involved bovine cerebral cortex membranes. Although the extracts contain catecholamines (brain only), histamine (lung only) and monovalent cations (both), which have the potential to interfere with the radioligand binding assays, their concentrations were too low to account for the effects observed. Preliminary attempts at purification of the extracts revealed that CDS activities from the two tissues are similar, i.e., practically insoluble in organic solvents at room temperature, not affected by either Sep-Pak C18 column or anion exchange resins but retained (along with the monovalent cations) by cation exchange resin. However, following chromatographic separation on a Biogel P2 column, the CDS-containing eluates are cation-free and exhibit qualitatively similar elution profiles. Future experiments will involve further purification of 'clonidine-displacing substance' to characterize its interaction with alpha 2-adrenoceptor binding sites in greater detail and establish whether it has biological activity consistent with the properties implied by its effects in radioligand binding assays.

Species-selective binding of [3H]-idazoxan to alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in the central nervous system.

1. We have used the imidazoline derivative [3H]-idazoxan to define alpha 2-adrenoceptors and non-adrenoceptor, imidazoline binding sites in cerebral cortex membranes of calf, mouse, rat, guinea-pig and man. 2. Competition experiments using the selective alpha-adrenoceptor drugs, rauwolscine and corynanthine, indicated that [3H]-idazoxan bound to a single population of sites in the calf and mouse membranes. However, [3H]-idazoxan also labelled non-adrenoceptor, imidazoline binding sites in the rat (15%), guinea-pig (30%) and human (40%) cerebral cortex membranes. 3. Competition experiments with adrenaline and cirazoline in the guinea-pig cortex, verified [3H]-idazoxan binding to both alpha 2-adrenoceptors and to non-adrenoceptor, imidazoline binding sites. 4. It has been postulated by several groups that [3H]-idazoxan may possess partial agonist activity. To investigate this further, saturation experiments were performed in the cerebral cortex membranes of all five species in the absence and presence of 300 microM guanosine triphosphate (GTP). GTP had no effect on [3H]-idazoxan binding in guinea-pig cerebral cortex; in both rat and mouse membranes 300 microM GTP increased the dissociation constant for [3H]-idazoxan by 2-3 fold without significantly affecting the Bmax. GTP reduced the Bmax by approximately 30% and 60% in calf and human cerebral cortex membranes, respectively, without significantly altering the Kd. 5. Saturation experiments were performed in the calf cerebral cortex membranes in the absence and presence of 300 microM GTP with the selective alpha 2-adrenoceptor agonist [3H]-clonidine and the selective muscarinic antagonist [3H]-quinuclidinyl benzilate (QNB). GTP reduced the Bmax for [3H]-clonidine without altering the Kd, but failed to affect either the Bmax or the Kd for [3H]-QNB.6. Saturation experiments were performed in human cerebral cortex membranes in the presence of alpha2-adrenoceptor blockade with and without GTP. GTP 300 microM reduced the Bmax for [3H]-idazoxan at the non-adrenoceptor, imidazoline binding sites, without affecting the Kd. GTP did not affect [3H]-QNB binding to muscarinic sites.7. Thus, there is a need to investigate further the pharmacological actions of [3H]-idazoxan in view of its ability to recognise both alpha2-adrenoceptors and non-adrenoceptor, imidazoline binding sites and because it might possess agonist activity at some of these sites.