Antigen selectivity characteristic of polyclonal antibodies against omega-conotoxin GVIA and N-type voltage-dependent calcium channels.

The antibodies against omega-conotoxin GVIA (omega-CTX GVIA; N-type voltage-dependent calcium channel [VDCC] blocker) and B1Nt (N-terminal segment [residues 1-13] of BI alpha1 subunits of VDCCs) were prepared, and the selectivity for each antigen omega-CTX GVIA and B1Nt was investigated. For the antigen selectivity of anti-omega-CTX GVIA antibody against omega-CTX GVIA, ELISA, and immunoprecipitation were used. The reactions for ELISA and immunoprecipitation were observed except when antibody IgG purified by Protein A-Sepharose CL-4B from nonimmunized serum (purified NI-Ab) was used. The specific reactions were inhibited by 10 nM omega-CTX GVIA, but not by omega-CTX SVIB (N-type VDCC blocker), omega-CTX MVIIC (N- and P-type VDCC blocker), or omega-Aga IVA (P-type VDCC blocker). For the antigen selectivity of the anti-B1Nt antibody, analyses by ELISA, immunoprecipitation, and Western blotting were conducted. The reactions were observed except when NI-Ab was used. The ELISA and immunoprecipitation reactions were inhibited by the antigen peptide B1Nt, and the IC50 values were about 1.2 x 10(-8) and 1.3 x 10(-8) M, respectively. The bands of 210 and 190 kD by Western blotting of crude membranes from chick brain were also inhibited by 1 microM B1Nt. These results suggest that the antibodies prepared against omega-CTX GVIA and B1Nt in this work have high selectivity for their antigen. Therefore we assume that the antibodies against omega-CTX GVIA and B1Nt are useful tools for the analyses of the function and distribution of N-type VDCCs. The anti omega-CTX GVIA antibody must also be useful for the radioimmunoassay of omega-CTX GVIA.

UT841 purified from sea urchin (Toxopneustes pileolus) venom inhibits time-dependent (45)Ca(2+) uptake in crude synaptosome fraction from chick brain.

To clarify the mechanism by which the toxic abstract from Toxopneustes pileolus inhibits time-dependent (Time-dep.) Ca(2+) uptake in crude synaptosome fraction, the effective component from pedicellarial venom of the sea urchin was purified. The crude extracts were purified by a series of steps including ion exchange (DEAE-sephadex-A25 gel), gel filtration (with Superdex-2000 and Superdex-peptide columns) and reversed-phase chromatography (Sephasil-C18 column). The effective component that inhibited Time-dep. 45Ca(2+) uptake was purified and named UT841. Its IC(50) was determined to be lower than 35ng/ml. UT841 is an acidic protein with an apparent molecular weight of about 18,000. The N-terminal sequence (40 amino acids) was almost identical to that of Contractin A (a protein purified from the same kind of venom which induces smooth muscle contraction). Even though it is unclear whether or not UT841 is Contractin A, Ca(2+) mobilization in nerve cells was shown to be influenced by UT841. This investigation also revealed that a donor of nitric oxide, arachidonic acid and an inhibitor of phospholipase C selectively inhibit Time-dep. (45)Ca(2+) uptake. These results suggest that UT841 purified from sea urchin venom may affect Time-dep. (45)Ca(2+) uptake through the metabolism of some lipids and nitric oxide.

Pancreatic exocrine damage induced by subcutaneous injection of a low dosage of zinc.

The aim of this study was to observe whether a low dosage of zinc induced mouse pancreatic injury. Dosages of zinc from 0.1 to 50 mg/kg were injected subcutaneously in mice, and plasma and pancreatic clinical parameters were observed 3-24 h after the injection. Plasma alpha-amylase activity increased 10 and 24 h after the injection of 25 or 50 mg/kg of zinc, whereas pancreatic alpha-amylase activity decreased 3 h after more than 5 mg/kg of zinc was injected. The activity recovered after 24 h except in the group injected with 50 mg/kg of zinc. The plasma glucose level did not change when less than 25 mg/kg of zinc was injected. The pancreatic zinc contents increased 3 h after more than 1 mg/kg of zinc was injected. The pancreatic metallothionein (MT) contents increased 6 h after the injection of 1 mg/kg of zinc. In addition, when more than 5 mg/kg of zinc was injected, the MT content increased at 3 h. In histochemical observations, cell damages such as fibrosis and necrosis were observed in pancreatic exocrine cells, but not in cells of Langerhans islets. From the present study, a single injection of a low dosage of zinc induces injury in pancreatic exocrine cells, but not endocrine cells.

Calcium/calmodulin inhibits the binding of specific [125I]omega-conotoxin GVIA to chick brain membranes.

The effect of Ca2+/calmodulin (CaM) on the specific binding of [125I]omega-conotoxin GVIA (125I-omega-CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [125I]omega-CTX binding to crude membranes, we observed that Ca2+/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca2+/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca2+ and CaM, and this effect was dependent on the concentration of Ca2+. The effect of Ca2+/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290-309). An experiment with modified ELISA using purified anti omega-CTX antibody indicated that Ca2+/CaM did not affect the direct binding of [125I]omega-CTX and CaM. These results suggest that Ca2+/CaM either directly or indirectly affects specific [125I]omega-CTX binding sites, probably N-type Ca2+ channels in crude membranes from chick whole brain.

Inhibitory effects of tetrahydroisoquinoline derivatives on Ca2+ and Na+ channels in crude nerve endings.

Semi-synthetic tetrahydroisoquinoline derivatives prepared from natural alkaloids, possess Ca2+ antagonistic properties. These derivatives significantly blocked KCl-stimulated Ca2+ uptake (In chick and rat crude nerve endings) which can be partially inhibited by the selective N-type Ca2+ channel blocker omega-conotoxin GVIA or the selective P-type Ca2+ channel blocker omega-agatoxin IVA. Moreover, PX42 (10 microM; for the tetrahydroisoquinoline compounds in this study) could inhibit the activity of calmodulin-dependent phosphodiesterase and block veratridine-induced (or tetrodotoxin-sensitive) Na+ uptake. The possible mechanism(s) of non-selective inhibition of ion channels of PX42 is discussed.

Characteristics of the inhibitory effect of calmodulin on specific [125i]omega-conotoxin GVIA binding to crude membranes from chick brain.

The characteristics of the inhibitory effect of calcium ion (Ca2+)/calmodulin (CaM) on specific [125I]-omega-conotoxin GVIA (125I-omega-CTX) binding and on the labeling of 125I-omega-CTX to crude membranes from chick brain were investigated. The inhibitory effect of Ca2+/CaM depended on the concentrations of free Ca2+ and CaM. The IC50 values for free Ca2+ and CaM were about 2.0 x 10(-8) M and 3.0 microg protein/ml, respectively. The inhibitory effect of Ca2+/CaM was attenuated by the CaM antagonists W-7, prenylamine and CaM-kinase II fragment (290-309), but not by the calcineurin inhibitor FK506. Ca2+/CaM also inhibited the labeling of a 135-kDa band (which was considered to be part of N-type Ca2+ channel alpha1 subunits) with 125I-omega-CTX using a cross-linker. These results suggest that Ca2+/CaM affects specific 125I-omega-CTX binding sites, probably N-type Ca2+ channel alpha1 subunits, in crude membranes from chick whole brain.

Proportions of Ca2+ channel subtypes in chick or rat P2 fraction and NG108-15 cells using various Ca2+ blockers.

The proportions of calcium (Ca2+) channel subtypes in chick or rat P2 fraction and NG 108-15 cells were investigated using selective L-, N-, P- and P/Q- type Ca2+ channel blockers. KCl-stimulated 45Ca2+ uptake by chick P2 fraction was blocked by 40-50% using N-type Ca2+ channel blockers [omega-conotoxin GVIA, aminoglycoside antibiotics and dynorphin A(1-13)], but was not inhibited by P- or P/Q-type blockers (omega-agatoxin IVA or omega-conotoxin MVIIC). On the other hand, KCl-stimulated 45Ca2+ uptake by rat P2 fraction was blocked by 30 approximately 40% using P- or P/Q-type Ca2+ channel blockers, but was not inhibited by N-type Ca2+ channel blockers. The L-type Ca2+ channel blockers 1,4-dihydropyridines, diltiazem and verapamil, but not calciseptine (CaS), inhibited both KCl-stimulated 45Ca2+ uptake and veratridine-induced 22Na+ uptake by chick or rat P2 fraction with similar IC50 values. CaS did not have any effect on 45Ca2+ uptake by either chick or rat P2 fraction. In NG108-15 cells, CaS, omega-agatoxin IVA and omega-conotoxin MVIIC, but not omega-conotoxin GVIA, inhibited KCl-stimulated 45Ca2+ uptake by 30-40%. Various combinations of these Ca2+ channel blockers had no significant additional effects in chick or rat P2 fraction or NG 108-15 cells. These findings suggest that KCl-stimulated 45Ca2+ uptake by chick or rat P2 fraction and NG 108-15 cells is a convenient and useful model for screening whether or not natural or synthetic substances have selective effects as L-, N-, P-, or P/Q- type Ca2+ channel antagonists or agonists.

A toxic extract from sea urchin (T. pileolus) inhibits 45Ca2+ uptake in P2 fraction from chick brain under physiological ionic conditions.

An acidic toxic extract from the sea urchin T. pileolus inhibited time-dependent 45Ca2+ uptake in chick P2 fraction (IC50 about 10 ng/ml). This toxic extract is susceptible to trypsin. The inhibitory effect probably relate to Na+/Ca2+ exchanger.

The inhibitory effect of the toxic fraction from sea urchin (Toxopneustes pileolus) venom on 45Ca2+ uptake in crude synaptosome fraction from chick brain.

The effects of toxic peaks (P-I, P-II and P-III eluted from Sephadex G-200 column) from the sea urchin Toxopneustes pileolus on time-dependent 45Ca2+ uptake in chick P2 fraction (crude synaptosome fraction) were studied under physiological ionic conditions. Time-dependent 45Ca2+ uptake was inhibited by P-II and P-III, but not by P-I. P-II had the greatest inhibitory effect. The inhibitory effect of P-II was not due to the inhibition of 45Ca2+ binding on P2 fraction, because P-II did not affect 45Ca2+ binding in osmotic-shocked P2 fraction. P-II did not affect KCl-stimulated 45Ca2+ uptake in P2 fraction, (Ca(2+)-Mg2+)-ATPase activity in the synaptic plasma membrane (SPM) fraction, or (Na(+)-K+)-ATPase and Mg(2+)-ATPase activities in osmotically-shocked P2 fraction. In contrast, the Na+/Ca2+ exchanger blocker 2',4'-dichlorobenzamil (DCB; 100 microM), with a poor specificity, inhibited not only time-dependent 45Ca2+ uptake but also KCl-stimulated 45Ca2+ uptake, (Ca(2+)-Mg2+)-ATPase, Mg(2+)-ATPase and (Na(+)-K+)-ATPase. Involvement of Na(+)-Ca2+ exchanger in the time-dependent 45Ca2+ uptake was ruled out, since it was not inhibited by replacement of Na+ with Li+ in reaction medium. These results suggested that the inhibition by P-II on time-dependent 45Ca2+ uptake appeared to be more specific than the commercially available Na+/Ca2+ exchanger blocker DCB, although the mechanism is not clear yet.

Relationship between specific binding of 125I-omega-conotoxin GVIA and GTP binding protein: effects of the GTP analogues, mastoparan and A1F4-.

We investigated whether the specific binding or labeling of 125I-omega-CgTX on crude membranes from chick whole brain was affected when endogenous GTP binding protein (G protein) was activated by GTP analogues, mastoparan (MP) and aluminum fluoride (AIF4-; AICl3 + NaF). Both GTPgammaS and Gpp(NH)p attenuated the inhibitory effect of selective N-type Ca channel inhibitors such as aminoglycoside antibiotics (AGs) or dynorphine (1-13)(Dyn) on specific 125I-omega-CgTX binding in a dose-dependent manner. On the other hand, the inhibitory effects of the divalent metal cations Cd2+, Co2+, Mg2+ and Mn2- on such binding were not attenuated by GTPgammaS. MP and AIF4- also attenuated the inhibitory effect of Neo on this binding similar to GTPgammaS. The attenuating effect of MP was enhanced by the presence of Mg2+ in a dose-dependent manner. However, GTP analogues, MP and AIF4-, did not affect binding or labeling without AGs or Dyn. GTPgammaS, MP and AIF4- also attenuated the specific labeling of a 215-kDa band in crude membranes with 125I-omega-CgTX using the cross-linker DSS (non-reduced condition) in the presence of Neo. These results indicate that there are direct or indirect relationships between N-type Ca channels and G proteins via binding sites for AGs or MP.

Binding and labeling of omega-conotoxin GVIA in crude membranes from subfractionated fractions and various areas of chick brain.

Specific binding and specific labeling of 125I-omega-CgTX were investigated in crude membranes from both subfractionated fractions and various brain areas in chick whole brain. The specific activities of the marker enzymes 2',3'-cyclic nucleotide 3'-phosphorylase, Na/K ATPase and succinic dehydrogenase in the subfractionated fractions were three- to five-fold higher than those in the P2 fraction. However, the amount of specific [125I] omega-CgTX binding in the fractions of synaptosomes and synaptic plasma membranes was only about 1.2-times higher than that in the P2 fraction. The characteristics of specific 125I-omega-CgTX labeling with disuccinimidyl suberate to the 135-kDa band were generally comparable to those of specific [125I] omega-CgTX binding sites. These results suggest that the specific binding sites of [125I] omega-CgTX were not localized the synaptosomes and synaptic plasma membranes fractions, although each fraction was well isolated from the others from which were decided by the strength of specific activity for marker enzymes.

Pathogenic significance of interleukin-6 in a patient with antiglomerular basement membrane antibody-induced glomerulonephritis with multinucleated giant cells.

We report a patient with anti-glomerular basement membrane disease who developed renal failure associated with systemic manifestations, including acute-phase inflammatory reactions and plasmacytosis. Renal tissue obtained by an open surgical biopsy showed circumferential cellular crescents, multinucleated giant cells, and exudation of fibrin in all glomeruli. Immunofluorescence microscopy demonstrated deposition of immunoglobulin G, C3, and membrane attack complex along glomerular capillary walls. Multinucleated giant cells were suggested to be macrophage-monocyte lineage because they were CD68 positive. Bone marrow aspiration showed an increase of plasma cells. Immunostaining showed intensive expression of interleukin-6 (IL-6) in practically every part of the renal sites involving multinucleated cells, crescents, tubules, and infiltrating cells, suggesting that one of the sources of systemically elevated IL-6 was the kidney. Serum IL-6, anti-glomerular basement membrane antibody, and acute-phase proteins were markedly elevated, and returned dramatically to the normal level after corticosteroid therapy and plasmapheresis. We believe that IL-6 played an important role in the development of many symptoms in the present case.

Characteristics of [125I]omega-conotoxin labeling using bifunctional cross linker DSP in crude membranes from chick brain.

Characteristic of [125I]omega-conotoxin (omega-CgTX) labeling using bifunctional cross linker (dithio bis[succinimidyl propionate]:DSP) was systematically investigated in crude membranes from chick whole brain. [125I]omega-CgTX specifically labeled 216 kDa as a main and 236 kDa as a minor bands in the crude membranes under non-reduced condition, but not labeled under reduced condition. We investigated the effect of various Ca channel antagonists on [125I]omega-CgTX labeling with DSP in detail, and found that there is a strong correlation between the effects of Ca channel antagonists on [125I]omega-CgTX labeling of the 216 kDa band and specific [125I]omega-CgTX binding. These results suggest that labeling of the 216 kDa band under non-reduced condition with [125I]omega-CgTX using DSP involves the specific binding sites of [125I]omega-CgTX, perhaps including one of the neuronal N-type Ca channel subunits in the crude membranes.

Characteristics of specific 125I-omega-conotoxin GVIA binding and 125I-omega-conotoxin GVIA labeling using bifunctional crosslinkers in crude membranes from chick whole brain.

Characteristics of specific 125I-omega-conotoxin GVIA (125I-omega-CgTX) binding and 125I-omega-CgTX labeling using bifunctional crosslinkers were systematically investigated in crude membranes from chick whole brain. Aminoglycosides and dynorphine A (1-13) inhibited the specific binding of 125I-omega-CgTX, but not that of the L-type calcium ion channel antagonist [3H](+)PN200-110. It seems likely that the inhibitory effect of dynorphine A (1-13) does not involve kappa-opiate receptors, based on results with the opiate receptor antagonist naloxone and the kappa-opiate receptor agonist U50488H. Spider venom, Cd2+ and La3+ inhibited the specific binding of 125I-omega-CgTX, as well as that of [3H](+)PN200-110. Various L-type Ca2+ channel antagonists did not affect the specific binding of 125I-omega-CgTX. 125I-omega-CgTX specifically labeled 135 kDa and 215 kDa bands in crude membranes under reduced and non-reduced conditions, respectively. The crosslinker disuccinimidyl suberate (DSS) yielded better 125I-omega-CgTX labeling than the other two crosslinkers tested. We investigated the effect of various Ca2+ channel antagonists on 125I-omega-CgTX labeling with DSS in detail, and found that there is a strong correlation between the effects of Ca2+ channel antagonists on 125I-omega-CgTX labeling of the 135 kDa band and specific 125I-omega-CgTX binding. These results suggest that aminoglycosides and dynorphine A (1-13) are specific inhibitors of specific 125I-omega-CgTX binding, and that labeling of the 135 kDa band with 125I-omega-CgTX using DSS involves the specific binding sites of 125I-omega-CgTX, perhaps including one of the neuronal N-type Ca2+ channel subunits in the crude membranes.

A case of renal sarcoidosis showing central necrosis and abnormal expression of angiotensin converting enzyme in the granuloma.

We describe a 66-year-old man who developed renal failure related to granulomatous renal sarcoidosis without systemic manifestations. Renal failure was severe enough to require hemodialysis transiently. Renal biopsy of this patient revealed the central necrosis of the granuloma which is usually absent in sarcoid granuloma. Serum level of angiotensin converting enzyme (ACE) was not helpful for diagnosis in this patient because serum ACE level is often elevated in the condition of chronic renal failure. Immunohistochemical detection of ACE was of diagnostic value in this patient. Subsequent course in which glucocorticoid was used for therapy was consistent with the diagnosis. This is the first report of identification of ACE in renal sarcoid granuloma.

In vivo effects of monoclonal antibodies that functionally inhibit complement regulatory proteins in rats.

The present work was designed to evaluate the effects of functional suppression of complement regulatory proteins in vivo. Male Wistar rats were anesthetized with Nembutal and were intravenously injected with 1 mg/kg of F(ab')2 or Fab fraction of either monoclonal antibody 5I2, which inhibits the function of rat counterpart of mouse Crry/p65, or monoclonal antibody 6D1, which inhibits the rat counterpart of CD59. Mean arterial pressure was continuously measured for 30 min. When 5I2 was injected, there was a biphasic change of mean arterial pressure, namely, the rapid increase immediately after the injection (approximately 2 min, phase 1) and the subsequent fall and slow recovery (approximately 4-30 min, phase 2). These effects were completely abrogated by pretreatment of rats with cobra venom factor. Pretreatment with carboxypeptidase inhibitor, which inhibits inactivation of anaphylatoxins C3a and C5a, induced enhanced reduction of blood pressure. Circulating leukocytes and platelets were rapidly decreased 5 min after antibody injection and became normal by 2 h. Hematocrit and erythrocyte count were continuously increased up to 2 h after injection, suggesting that there was hemoconcentration due to increased vascular permeability. Immunofluorescence study revealed binding of antibody fragments and rat C3 along the capillaries of lung, heart, and liver 5 min after injection. In contrast to 5I2, F(ab')2 fraction of 6D1, though localized to the same areas and in similar amounts, had no significant effect on the parameters measured. These data suggest that the rat counterpart of mouse Crry/p65 plays a vital role in vivo by preventing the activation of autologous complement on vascular endothelium.

Localization of the complement regulatory proteins in the normal human kidney.

The kidney is an organ where complement-mediated tissue injuries take place by various stimuli. To assess how the kidney is protected from the autologous complement attack, comparative localization of decay accelerating factor (DAF), membrane cofactor protein (MCP) and 20 kDa homologous restriction factor (HRF20) was studied in the normal human kidney. Specific monoclonal antibodies to DAF, MCP and HRF20 were used for the study. Studies by immunofluorescence and immunoelectron microscopy showed that the distribution of each protein in the kidney was complementary to each other in most parts. MCP and HRF20 were clearly seen in the glomerular capillaries, while DAF was only faintly observed. Juxtaglomerular apparatus was abundant in DAF and MCP but not in HRF20. HRF20 was most strongly expressed in the peritubular capillaries where MCP was not detectable. Basolateral membranes of the proximal tubules and collecting ducts expressed MCP strongly, while there was no expression of DAF in the proximal tubules. Interestingly, both DAF and MCP, which inhibit complement activation at C3/C4 level, were not expressed in the apical portion of the tubular cells including proximal tubule brush border. In contrast, HRF20 was expressed on the apical part of the tubules. Medullary interstitium strongly expressed MCP but not DAF. Based on these observations, we conclude that each segment of the kidney is protected from the complement attack by the different combination of complement regulatory proteins. We speculate that the tubular cells might be fragile when complements are activated inside the tubular lumen, because there is no expression of complement regulatory proteins which inhibit C3 convertase.

Effects of Ca channel agonists on 45Ca uptake differ depending on the state of NG108-15 cells.

We investigated the effects of various Ca channel agonists (Ca agonist) derived from 1,4-dihydropyridine on KCl-stimulated 45Ca uptake by differentiated and undifferentiated neuroblastoma x glioma hybrid NG108-15 cells with and without dibutyryl cAMP. Ca agonists Bay K 8644, YC-170 and CGP 28392 enhanced KCl-stimulated 45Ca uptake in differentiated NG108-15 cells, but only slightly in undifferentiated NG108-15 cells. The rank order of the enhancing effects was roughly Bay K 8644 > YC-170 > > CGP 28392. These results suggest that there is some difference between the mechanism by which these Ca agonists affect KCl-stimulated 45Ca uptake in differentiated and undifferentiated NG108-15 cells, although the nature of that difference is not clear.

Characteristics of specific 125I-omega-conotoxin GVIA binding in rat whole brain.

Characteristics of specific 125I-omega-conotoxin (omega-CgTX) binding were systematically investigated in crude membranes from rat whole brain. Kd and Bmax Values for the binding were 49.7 pM and 181.5 fmol/mg of protein, respectively. The effects of various types of Ca channel antagonists on the binding were investigated. Dynorphin A (1-13), in particular, specifically inhibited 125I-omega-CgTX binding, but not that of [3H](+)PN200-110. Spider venom from Plectreurys tristes did not specifically inhibit specific binding of 125I-omega-CgTX, because the venom also inhibited the binding of [3H](+)PN200-110 to a similar degree. The amount of specific binding of 125I-omega-CgTX was less in the cerebellum than that in any other area of whole brain. The cross-linker disuccinimidyl suberate did not label with 125I-omega-CgTX and its binding sites in rat whole brain, although it did in chick whole brain, which was used as a positive control. These findings suggested that dynorphine A (1-13) was a selective blocker of omega-CgTX-sensitive Ca channels in crude membranes from rat whole brain and that omega-CgTX-sensitive Ca channels were mainly present a rat brain except cerebellum.

Specific bindings of [3H](+)PN200-110 and [125I]omega-conotoxin to crude membranes from differentiated NG108-15 cells.

The characteristics of the specific bindings of [3H](+)PN200-110 (PN: L-type Ca channel antagonist) and [125I]omega-conotoxin G VI A (omega-CgTX: neuronal L- or N-type Ca channel antagonist) to crude membranes from undifferentiated neuroblastoma X glioma hybrid NG108-15 (NG108-15) cells and differentiated cells induced with dibutyryl cAMP (Bt2cAMP) were examined, because we have already observed that the magnitude and rate of KCl-stimulated 45Ca uptake by NG108-15 cells increased progressively during differentiation of the cells induced with Bt2-cAMP (unpublished results). The specific binding of [3H](+)PN to these crude membranes was saturable at various concentrations of 2.5-5.0 nM [3H](+)PN. Scatchard analysis showed that the specific binding of [3H](+)PN at equilibrium was significantly increased after differentiation of the NG108-15 cells with Bt2cAMP, but that the apparent Kd value for the specific binding of [3H](+)PN was not influenced by treatment with Bt2cAMP. The specific binding of [3H](+)PN to crude membranes from Bt2cAMP-treated NG108-15 cells was inhibited by a calcium agonist and antagonists, the order of their inhibitory potencies being (+)PN > nitrendipine > (-)PN > or = Bay K 8644 > > diltiazem = verapamil. Thus, PNs showed significant stereoselective inhibition of the specific binding of [3H](+)PN. On the other hand, [125I]omega-CgTX at concentrations of 0.075-0.6 nM showed scarcely any specific binding to these crude membranes, although at 0.6 nM it showed specific binding to crude membranes from rat brain in the same experimental conditions.(ABSTRACT TRUNCATED AT 250 WORDS)