Bruce D. Cherksey

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.

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.

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.

Pharmacological characterization of the voltage-dependent Ca2+ channels present in synaptosomes from rat and chicken central nervous system.

Abstract: The voltage‐dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L‐type channels (1,4‐dihydropyridines), N‐type channels (ω‐conotoxin GVIA), and P‐type channels [funnel web toxin (FTX) and ω‐agatoxin IVA]. K+‐induced Ca2+ uptake by chicken synaptosomes was blocked by ω‐conotoxin GVIA (IC50 = 250 nM). This toxin at 5 µM did not block Ca2+ entry into rat frontal cortex synaptosomes. FTX and ω‐agatoxin IVA blocked Ca2+ uptake by rat synaptosomes (IC50 = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω‐agatoxin IVA affected Ca2+ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC50 similar to the one obtained in rat preparations, whereas with ω‐agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω‐conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω‐conotoxin GVIA was never greater than the one observed with ω‐conotoxin GVIA. We also found that 60% of the Ca2+ uptake by rat and chicken synaptosomes was inhibited by ω‐conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N‐type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca2+ uptake in either the rat or the chicken. In conclusion, Ca2+ uptake by rat synaptosomes is potently inhibited by different P‐type Ca2+ channel blockers, thus indicating that P‐type channels are predominant in this preparation. In contrast, Ca2+ uptake by chicken synaptosomes is sensitive to ω‐conotoxin GVIA, FTX, ω‐agatoxin IVA, and ω‐conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.

Tolbutamide and glyburide differ in effectiveness to displace α- and β-adrenergic radioligands in pancreatic islet cells and membranes

Previous in vivo findings indicated that α-adrenergic blocking agents enhanced tolbutamide-induced insulin secretion, whereas β-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 α- and β-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 α- and β-radioligands but had a higher affinity at the β-receptor. Glyburide also displaced radioligands from both types of receptors but had a higher affinity for the α-receptor. This study suggests that these two sulfonylurea hypoglycemic agents may affect insulin secretion by different mechanisms.

Adrenergic regulation of chloride secretion across the opercular epithelium: The role of cyclic AMP

SummaryActivation of the β-adrenergic receptors of the opercular epithelium ofFundulus heteroclitus stimulates Cl− secretion, while activation of the α-adrenergic receptors inhibits Cl− secretion (Degnan and Zadunaisky, 1979). The possible involvement of adenosine 3′, 5′-monophosphate (cAMP) in these adrenergic responses was investigated. Isolated opercular epithelia incubated in Ringer, containing 10 mM theophylline, had cAMP levels ranging between 5.3 and 19.3 pmoles·mg protein−1 (mean=9.5±1.0 pmoles·mg protein−1). Activation of the β-receptors by 10−5 M isoproterenol increased the mean cAMP level 430% (P<0.001). Blockage of the β-receptors with propranolol greatly reduced the increase in cAMP in response to isoproterenol. Activation of the α-receptors by 10−5 M arterenol stimulated the mean cAMP level 270% (P<0.01). However, when the β-receptors were blocked with propranolol, arterenol had no effect on the cAMP level. The possible involvement of Ca++ in these adrenergic responses was investigated. Neither the stimulatory effect of isoproterenol, nor the inhibitory effect of arterenol on the Cl− secretion were diminished in the absence of extracellular Ca++. The Ca++ ionophore, A23187, and the calmodulin inhibitor, trifluoperazine, had no effects on the Cl− secretion. The Ca++-channel blocker, D600, had a significant inhibitory effect (P<0.005). Guanosine 3′,5′-monophosphate (cGMP) had no effect on the Cl− secretion.The results indicate that β-adrenergic stimulation of Cl− secretion across the opercular epithelium is accompanied by an elevation in tissue cAMP levels. α-adrenergic inhibition of Cl− secretion does not involve changes in the tissue cAMP. Neither of these responses appear to require Ca++.

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.

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.

Isolation and reconstitution of furosemide-binding proteins from Ehrlich ascites tumor cells.

SummaryFurosemide-binding proteins were isolated from cholate-solubilized membranes of Ehrlich ascites tumor cells by affinity chromatography, using furosemide as ligand. Solubilized proteins retarded by the affinity material were eluted by furosemide. In reducing and denaturing gels, the major proteins eluted by furosemide were 100 and 45 kDa. In nonreducing, nondenaturing gels, homodimers of both polypeptides were found, whereas no oligomeric proteins containing both polypeptides were seen. It is concluded that the furosemide gel binds two distinct dimeric proteins. The isolated proteins were reconstituted into phospholipid vesicles and the K+ transport activity of these vesicles was assayed by measurement of86Rb+ uptake against a large opposing K+ gradient. The reconstituted system was found to contain a K+ transporting protein, which is sensitive to Ba2+ like the K+ channel previously demonstrated to be activated in intact cells after cell swelling.

Demonstration of. cap alpha. /sub 2/-adrenergic receptors in rat pancreatic islets using radioligand binding

Abstract The type of the α-adrenergic receptors on rat pancreatic islet cells was characterized directly using specific radioligands and displacement agonists and antagonists. Scatchard plots for binding of [3H]clonidine (α2-agonist) revealed a dissociation constant, K d of 0.552 ± 0.1 nM and density of binding sites (Bmax) of 50.4 ± 3.6 fmole/mg protein. Similar values were obtained with [3H]dihydroergocryptine (antagonist). The various agonists displaced [3H]clonidine with the following order of potency: clonidine > epinephrine > norepinephrine > isoproterenol. Yohimbine, the α2-antagonist, was very effective in displacing [3H]clonidine, whereas the α1-antagonist, prazosin, was much less effective. The data indicate that the α-adrenergic receptors on rat pancreatic islets are of the α2 subtype.