P-type calcium channels in the somata and dendrites of adult cerebellar Purkinje cells.

The pharmacological and single-channel properties of Ca2+ channels were studied in the somata and dendrites of adult cerebellar Purkinje cells. The Ca2+ channels were exclusively of the high threshold type: low threshold Ca2+ channels were not found. These high threshold channels were not blocked by omega-conotoxin GVIA and were inhibited rather than activated by BAY K 8644. They were therefore pharmacologically distinct from high threshold N- and L-type channels. Funnel web spider toxin was an effective blocker. The channels opened to conductance levels of 9, 14, and 19 pS (in 110 mM Ba2+). These slope conductances were in the range of those reported for N- and L-type channels. Our results are in agreement with previous reports suggesting that Ca2+ channels in Purkinje cells can be classified as P-type channels according to their pharmacology. The results also suggest that distinctions among Ca2+ channel types based on the single-channel conductance are not definitive.

Distribution and functional significance of the P-type, voltage-dependent Ca2+ channels in the mammalian central nervous system.

In addition to the three types of voltage-dependent calcium channels presently recognized in the CNS, the L-, the T- and the N-types, a fourth distinct type known as the P-type channel has recently been described. This channel, initially recognized in Purkinje cells (and thus the name), is not blocked by dihydropyridines or by omega-conotoxin (GVIA), but is blocked by native funnel-web spider venom and by a polyamine (FTX) extracted from such venom. In addition, a synthetic polyamine (sFTX) has been produced that also specifically blocks P-channels in brain slices and at the neuromuscular junction, and blocks presynaptic Ca2+ currents in other vertebrate and invertebrate forms, as well as channels expressed in Xenopus oocytes following CNS mRNA injections. Using sFTX to form an affinity gel, a protein was isolated and reconstituted into lipid bilayers where it manifests single-channel properties that are electrophysiologically and pharmacologically similar to those of the native P-channels. Rabbits immunized with the isolated protein produced a polyclonal antibody that gave a positive western blot with the purified P-channel protein and generated a reaction product at specific sites in the CNS that agree with the physiological distribution of P-channel activity.

Tolbutamide and glyburide differ in effectiveness to displace alpha- and beta-adrenergic radioligands in pancreatic islet cells and membranes.

Previous in vivo findings indicated that alpha-adrenergic blocking agents enhanced tolbutamide-induced insulin secretion, whereas beta-blockade attenuated it. In the present study, the interaction of tolbutamide and glyburide with the rat islet adrenergic receptors is examined directly by determining the effectiveness of these drugs to displace the specific alpha- and beta-adrenergic radioligands, [3H]-clonidine and [3H]-dihydroalprenolol (DHA). It was found that both tolbutamide and glyburide had affinity constants for the adrenergic receptors that were similar to those for the natural receptor ligands and powerful antagonists. Tolbutamide displaced both alpha- and beta-radioligands but had a higher affinity at the beta-receptor. Glyburide also displaced radioligands from both types of receptors but had a higher affinity for the alpha-receptor. This study suggests that these two sulfonylurea hypoglycemic agents may affect insulin secretion by different mechanisms.

Preponderance of beta-adrenergic binding sites in pancreatic islet cells of the rat.

Although the alpha- and beta-adrenergic receptors have opposing effects on insulin secretion, the inhibitory influence of alpha-receptors appears to predominate. To determine if this was due to differences in number and affinity of receptors, isolated rat pancreatic islet cells were incubated with [3H]-dihydroalprenolol and [3H]-dihydroergocryptine as ligands for beta- and alpha-adrenergic binding sites. It was found that the number of beta-adrenergic binding sites was 143 fmol/mg islet protein with a Kd = 0.57 nM. The number of alpha-adrenergic binding sites was 53 fmol/mg protein with a Kd = 0.26 nM. Thus, there are 2.7 times as many beta-adrenergic binding sites as alpha-binding sites, and neither binding site number nor affinity is responsible for the predominant influence of the alpha-adrenergic receptors.

Bipolar mood disorder and endometriosis: preliminary findings.

A consecutive sample of 16 women with laparoscopy-diagnosed endometriosis were evaluated for mood disorders. Twelve women met DSM-III criteria for a mood disorder: seven for bipolar disorder, mixed, three for bipolar disorder, manic, and two for major depression. Two women had equivocal diagnoses and two showed no evidence of mood disorder. Nine subjects had first-degree relatives with histories of severe mood disorders.

Adrenal chromaffin cells on microcarriers exhibit enhanced long-term functional effects when implanted into the mammalian brain.

Rat adrenal chromaffin cells attached to either collagen-coated dextran (Cytodex 3) or glass bead microcarriers, both of 90-200 microns diameter, were used as dopamine-secreting implants in the caudate-putamen of rats with 6-hydroxydopamine-induced unilateral lesions of the substantia nigra. As controls, beads without cells and cells in suspension alone were implanted. Chromaffin cells adhered to microcarriers reduced apomorphine-induced rotation by 75% in lesioned animals. Animals that were lesioned but not receiving cell implants or receiving beads alone showed no reduction. Animals implanted with cells not attached to beads also showed a reduction in rotation but this effect lasted less than three months. Microcarrier-attached cells, however, maintained their effect in reducing rotation for at least eight months (rotations were reduced from a control mean of 10.9 +/- 1.4 to 3.6 +/- 1.1 turns/min) without any "drop-off" of the effect. Histological examination showed that eight months post-implant the cells pre-adhered to beads were still present and could be stained by anti-tyrosine hydroxylase antibody. Sections stained with hematoxylin-eosin showed no signs of an inflammatory response. In contrast to beads implanted into the striatum, Cytodex bead implants injected into the lateral ventricle induced a histopathological response appearing to involve the ependyma and choroid plexus. Results suggest that the striatal parenchyma but not the ventricle is amenable to studies using the microcarrier approach to transplantation.

Different calcium channels mediate transmitter release evoked by transient or sustained depolarization at mammalian sympathetic ganglia.

We have compared the effect of calcium channel blockers on the potassium-evoked release of tritium-labeled acetylcholine and on preganglionic spike-evoked synaptic transmission in the rat superior cervical ganglion. Transmitter release at the nerve terminals is mediated by the influx of calcium through voltage-gated calcium channels. While four types of voltage-gated calcium channels (T, L, N and P) have been identified in neurons, it is not clear which may actually be involved in excitation-secretion coupling. Release of tritiated acetylcholine evoked by sustained depolarization in high (40 mM) extracellular potassium decreased markedly in the absence of calcium or the presence of cadmium. High potassium-evoked release was substantially inhibited by the P-type channel blockers, purified from funnel-web spider toxin, and omega-agatoxin-IVA, and by the N-type channel blocker omega-conotoxin-GVIA, but was unaffected by the L-type channel blocker nitrendipine. In contrast, postganglionic compound action potentials synaptically triggered by preganglionic stimulation were strongly blocked by funnel-web spider toxin and slightly blocked by a high concentration of omega-agatoxin-IVA, but were unaffected by either omega-conotoxin-GVIA, nitrendipine or a low concentration of omega-agatoxin-IVA. Thus, at the superior cervical ganglion, funnel-web spider toxin-sensitive calcium channels play a dominant role in transmitter release evoked by transient, spike-mediated depolarization, but other types of voltage-gated calcium channels in addition to the funnel-web spider toxin-sensitive channel mediate the transmitter release that is evoked by sustained high potassium depolarization.

Nonsteroidal antiinflammatory drugs exert differential effects on neutrophil function and plasma membrane viscosity. Studies in human neutrophils and liposomes.

Nonsteroidal antiinflammatory drugs (NSAIDs) inhibit neutrophil functions via mechanisms separate from their capacity to inhibit prostaglandin synthesis. We have studied discrete events in the process of signal transduction: NSAIDs but not a related analgesic drug (acetaminophen), inhibited aggregation in response to the chemoattractants f-Met-Leu-Phe (FMLP), leukotriene B4, and C5a. NSAIDs, but not acetaminophen, inhibited binding of radiolabeled FMLP to purified neutrophil membranes. Gpp(NH)p, a GTPase insensitive analog of GTP, also inhibited the binding of FMLP but, paradoxically, enhanced superoxide anion generation and lysozyme release. The inhibition of ligand binding by NSAIDs did not correlate with their capacity to inhibit FMLP-induced increments in diacylglycerol (DG): piroxicam, but not salicylate effectively inhibited appearance of label ([3H]arachidonate, [14C]glycerol) in DG. Finally, NSAIDs exerted differential effects on the viscosity of neutrophil plasma membranes and multilamellar vesicles (liposomes): membrane viscosity was increased by piroxicam and indomethacin, decreased by salicylate, and unaffected by acetaminophen. Thus, the different effects of NSAIDs on discrete pathways are not due to their shared capacity to reduce ligand binding but rather to a capacity to uncouple postreceptor signaling events that depend upon the state of membrane fluidity.

Functional reconstitution of an isolated sodium channel from bovine trachea.

An amiloride-sensitive Na+ channel from bovine trachea was isolated using an affinity gel and reconstituted into a planar lipid bilayer. This channel exhibited: 1. Fluctuations with long duration opening and closing times, weak voltage dependence, and a conductance of 6 pS. 2. Selectivity of at least 100-fold for Na+ over K+. 3. Saturates at a Na+ concentration of 90 mM. 4. Blocked by amiloride, 50% inhibition at 0.1 microM.

On the mechanism of potentiation by morphine of thiopental sleeping time.

Injection of thiopental sodium, 25 or 40 mg/kg, i.p., into normal rats produced a prompt sleep which lasted 8.3 plus or minus 2.9 and 16.8 plus or minus 2.7 min, respectively. Injection of morphine sulphage, 20 mg/kg, i.p., itself produced only mild sedation but prolonged the thiopental induced sleep to 50 and 67.3 min, respectively. At 1 min after onset of sleep the brain thiopental concentration in morphine pretreated animals was significantly lower than in control animals. Furthermore the latter also awoke with higher brain thiopental concentrations; the morphine pretreated animals slept for a longer time despite lower brain thiopental concentration. The t1/2 of brain thiopental was longer in the morphine pretreated animals, 25 min, than in the control animals, 9 min. Distribution of thiopental in several tissues at various time periods following injection of thiopental revealed some time-related quantitative differences between the two groups but a similar qualitative pattern. It is suggested that morphine lowers the brain threshold for thiopental induced sleep.

Electrodiffusion of Cl- and K+ in epithelial membranes reconstituted into planar lipid bilayers.

An electrodiffusive permeability for Cl-, its activation by low extracellular Cl--concentrations and the interaction between electrodiffusive fluxes of Cl- and K+ are demonstrated in the ventricular membranes from the epithelium of the bovine choroid plexus. Membranes were fused into artificial lipid bilayers formed at the tip of micropipettes. What is thought to be the cytoplasmic side of the membrane (the trans-side or the inside of pipette) was clamped at negative potentials (0 to -90 mV). Under these conditions the current was discrete, fluctuating less than 2 pA. With Cl- as the only conducting ion on the two sides we observed a small electrodiffusive permeability which was reduced by bumetanide or furosemide by 62%. When the outside solution was rendered Cl--free then the permeability to Cl- increased by a factor of 2-5; this activation was reduced by bumetanide or furosemide by about 80%. We observed an interaction between inwards movements of K+ and outwards movements of Cl- via the activated permeability: The total current was smaller than the sum of the expected inward K+-current and the expected outward activated Cl--current. Bumetanide or furosemide increased the total current; apparently the loss of current carried by Cl- was smaller than the gain in current carried by K+. The presence of K+ on both sides of the membrane was a condition for this interaction.

Cytoskeletal constraint of the beta-adrenergic receptor in frog erythrocyte membranes.

A fluorescence receptor binding assay, based upon the high-affinity beta-adrenergic receptor antagonist propranolol, is utilized to probe the microenvironment of the antagonist-receptor complex in the frog (Rana catesbeiana) erythrocyte membrane. The technique of steady-state fluorescence depolarization is applied to the propranolol-receptor complex, allowing quantitation of the rotational relaxation time of the complex. It is found that the complex is dynamically constrained at 20 degrees C. However, in the temperature range 6-10 degrees C a sharp reversible release of constraint is observed. It is further demonstrated that the addition of drugs that are known to specifically disrupt the cytoskeleton (colchicine, vincristine, and vinblastine) causes a similar but irreversible release of constraint at 20 degrees C. Cytochalasin B has a much smaller influence on the rotational mobility of the propranolol-receptor complex than do the other drugs that disrupt the cytoskeleton. Amphotericin B is without effect on the rotational constraint of the complex. Binding of the antagonist [3H]dihydroalprenolol is not influenced by colchicine. A model is proposed which postulates that cytoskeletal elements are linked to the antagonist-receptor complex. Antagonist binding does not result in cytoskeletal release, whereas agonist binding is postulated to lead to dissociation of the agonist-receptor complex from the cytoskeleton, thereby activating adenylate cyclase.

Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison.

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells.

Displacement of alpha- and beta-radioligands by specific adrenergic agonists in rat pancreatic islets.

Secretion of insulin is increased by beta-adrenergic agonists and inhibited by alpha-adrenergic agonists. However, administration of epinephrine, which acts on both types of receptors, inhibits insulin secretion. A preliminary study using [3H]-dihydroergocryptine and [3H]-dihydroalprenolol as the respective alpha- and beta-receptor binding ligands, surprisingly revealed a preponderance of beta-binding sites in normal rat pancreatic islets. The present study, using displacement by epinephrine, norepinephrine, isoproterenol and clonidine validated the use of these radioligands as appropriate for specific receptor binding in pancreatic islet cells. The islets were found to have 55 fmol/mg protein of alpha-adrenergic receptor sites and 170 fmol/mg protein of beta-receptor sites. The affinity of both alpha- and beta-receptors for epinephrine was similar, as judged by the displacement of either radioligand, thus ruling out a preferential affinity of alpha-receptor binding as an explanation for the alpha-inhibition of insulin secretion. The data on radioligand displacement by clonidine indicate that the alpha-receptor is of the alpha 2-type.

Fluorescence studies of the beta-adrenergic receptor topology.

The nature of the propranolol binding site of the beta-adrenergic receptor has been examined by utilizing the intrinsic fluorescence of propranolol as a probe. Additionally, the spatial relationship between the propranolol binding site and membrane tryptophan has been examined by utilizing I-quenching of intrinsic tryptophan fluorescence, chemical modification of membrane tryptophan, and singlet-singlet energy transfer between membrane-bound propranolol and tryptophan. Propranolol, at concentrations consistent with specific beta-receptor binding, protected approximately 42% of the membrane tryptophan fluorescence from I-quenching. Further, in the presence of propranolol, the apparent quenching constant (kq) was altered from 3.6 to 21.8 M-1. Reaction of the membrane fragments with 2-hydroxy-5-nitrobenzyl bromide (Koshland's reagent I) in the presence and absence of propranolol indicated that low concentrations of propranolol protected approximately 45% of the membrane tryptophan from the reagent. The singlet-singlet energy transfer from tryptophan to propranolol was determined by sensitized emission. The distance between these two species was found to be less than 20 A. These results have been interpreted to indicate that propranolol, when bound to the beta-adrenergic receptor, is situated such that its naphthyl moiety is inserted into a tryptophan-rich hydrophobic pocket of the receptor.