Isoenzymes of γ-glutamyl transpeptidase in liver diseases

Summary A new method for the separation of isoenzymes of-glutamyl-transpeptidase is described, using electrophoresis on acetate cellulose gel and a developing solution composed by-glutamyl-naphthylamide, and a colored diazonium compound.The method permits the separation of up to four different isoenzymes, which we called-GT₁,-GT₂,-GT₃,-GT₄, the first two showing an electrophoretic migration similar to that of ₁- and ₂-globulins and the other two to that of-globulins.The present technique has proved its usefulness in detecting isoenzymes in serum with values of total-glutamyl-transpeptidase higher than 80 U/L.The application of this method in 52 patients with different types of biliary obstruction and hepatocellular damage has shown that it provides new possibilities in differential diagnosis.     

Flow Cytometry Evaluation of the T-Cell ReceptorVβ Repertoire Among HIV-1 Infected Individuals Before and After Antiretroviral Therapy

HIV-1 infection leads to serious impairment of the immune system and perturbations in the T cell receptor V repertoire are also described. Immune reconstitution can be potentially achieved in response to HAART. In the present study 10 patients were investigated for the V pattern expression before and after six months of HAART. TCR were analyzed for T CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 24 different V chains. Compared to eight Brazilian healthy controls, no differences in V pattern of expression was observed for patients before or on antiretroviral therapy. Some chains such as V 3, 14, 16, 20 and 21.3 were over utilized by both T subsets, independently of HIV infection and/or antiretroviral treatment, differing from the ones described for individuals of other nationalities. However, when each patient was taken individually, particular alterations were detected for the V gene usage, compared to controls, for all individuals. After treatment, significant V usage changes were observed for seven patients. One or more chains on both T subsets were engaged in this process, defining a preferential oligoclonal profile for TCR repertoire distribution, after HAART. Although no pattern of specific V changes was detected in the circulating T cells, we cannot exclude that differential immune responses to HIV or other important antigens are being focused by these cells.     

Overshoot in <Emphasis Type="Italic">V̇</Emphasis>O<Subscript>2</Subscript> following the onset of moderate-intensity cycle exercise in trained cyclists

We have previously observed that following the onset of moderate intensity cycle ergometry, the pulmonary O₂ uptake (O₂) in trained cyclists often does not increase towards its steady-state value with the typical mono-exponential characteristics; rather, there is a transient overshoot. The purpose of this study was to systematically examine this phenomenon by comparing the O₂responses to two moderate-intensity work rates and one high-intensity work rate in trained and untrained subjects. Following a ramp exercise test to the limit of tolerance for the determination of the gas exchange threshold (GET) and O_2peak, seven trained cyclists [mean (SD); O_2peak 66.6 (2.5) ml·kg^–1·min^–1] and eight sedentary subjects [O_2peak 42.9 (5.1) ml·kg^–1·min^–1] completed six step transitions from baseline cycling to work rates requiring 60% and 80% GET and three step transitions from baseline cycling to a work rate requiring 50% of the difference between GET and O_2peak (50%). O₂ was measured breath-by-breath and modelled using standard techniques. The sedentary subjects did not overshoot the steady-state O₂ at any intensity. At 60% GET, six of the seven cyclists overshot the steady-state O₂ [by an integral volume of 164 (44) ml between ~45 and 125 s]. At 80% GET, four of the seven cyclists overshot the steady-state O₂ [by an integral volume of 185 (92) ml between ~55 and 140 s]. None of the cyclists showed an overshoot at 50%. These results indicate that trained cyclists evidence an overshoot in O₂ before steady-state is reached in the transition to moderate-intensity exercise. The mechanism(s) responsible for this effect remains to be elucidated, as does whether the overshoot confers any functional or performance benefit to the trained cyclist.     

Trennung der intestinalen β-Galactosidasen beim lactose-toleranten Erwachsenen durch Ultrazentrifugation im Dichtegradienten

Zusammenfassung Die intestinalen-Galactosidasen von 4 lactose-toleranten, erwachsenen Mitteleuropäern wurden im Saugbiopsie-Gewebe nach Solubilisierung mit Triton X-100 in einem linearen Mannitol-Gradienten (5–20%) auf der Ultrazentrifuge bei 4°C und 44000 U/min getrennt. Bei 12stündiger Zentrifugation fanden sich 3 Fraktionen, von denen die beiden schneller sedimenticrenden Lactose spalten. Alle 3 Fraktionen hydrolysieren p-Nitrophenyl--Galactosid. Die 3 isolierten-Galactosidasen entsprechen wahrscheinlich der neutralen Bürstensaum-Lactase, der sauren lysosomalen Lactase und einer cytoplasmatischen Hetero--Galactosidase.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Calcium overload toxicity and functional impairment in peritoneal leukocytes elicited by glycogen or interleukin-1β

Although calcium plays an important role in the activation of leukocytes for such processes as eicosanoid biosynthesis, secretion of granular constituents and superoxide generation, sustained high levels of intracellular calcium ions may be toxic. We have previously found that high concentrations of calcium ionophores induce a rapid-onset calcium overload toxicity in rat peritoneal leukocytes, in which functional responses such as -glucuronidase secretion, superoxide generation and leukotriene B₄ synthesis are greatly attenuated, and some leakage of cytoplasmic LDH occurs. We have now compared this phenomenon in peritoneal leukocytes elicited from animals pretreated in three ways: glycogen, interleukin-1 (IL-1) alone or glycogen plus IL-1. Peritoneal administration of IL-1 caused elicitation of cells which were enriched in eosinophils; however, the functional responses of the cells in all three groups were broadly similar in terms of the ability of the agonists FMLP, PMA and A23187 to initiate superoxide generation, -glucuronidase secretion and leukotriene generation. Cells from all three treatment groups showed diminished responsiveness at 10^–5 M A23187, indicative of calcium overload toxicity. This was most evident for the superoxide and -glucuronidase responses. Some quantitative differences observed between treatment groups may reflect the different sensitivities of the various cells contained in the mixed leukocyte preparations. We conclude that IL-1 induces leukocyte emigration into the peritoneal cavity but that the cell population is different from that induced by glycogen. However, the cells retain susceptibility to calcium overload toxicity.     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Differential induction of human monocyte transforming growth factorβ1 production and its regulation by interleukin 4

We have previously shown that trauma patients' monocytes which arein vivo activated by multiple injury-induced mediators have elevated transforming growth factor-beta (TGF) bioactivity. Interleukin-4 (IL-4), a Th₂ and B lymphocyte stimulatory factor, has been shown to inhibit monocyte production of a number of mediators both after lipopolysaccharide stimulation and after trauma-induced stimulation. However, IL-4 inhibitory effects appears to vary, depending on the mixture of inducing stimuli. Here we describe thein vitro IL-4 inhibition of human monocyte TGF bioactivity using several stimulation induction protocols: muramyl dipeptide stimulation alone, or after FcRI (CD64) cross-linking induction, interferon-gamma (IFN) priming, or trauma-generatedin vivo mediator induction. IL-4 suppressed both muramyl dipeptide-induced TGF bioactivity and TGF mRNA in a dose-dependent fashion and was most effective when IL-4 was administered at initiation of normal monocyte stimulation. Muramyl dipeptide (MDP)-induced increases in trauma patients' monocyte TGF bioactivity were also inhibited by high doses of IL-4 (25 ng/ml). FcRI cross-linking increased MDP-induced normal monocyte TGF bioactivity, but this increase could be consistently inhibited only by very high IL-4 concentrations (50 ng/ml). IL-4 did not consistently downregulate MDP-induced TGF bioactivity in IFN-primed monocytes. IL-4 can suppress monocyte TGF production, as well as other monocyte mediators, but its efficiency depends on the stimuli combination present in the microenvironment.     

Characterization of two components of the N-like,high-threshold-activated calcium channel current in differentiated SH-SY5Y cells

Retinoic acid differentiated SH-SY5Y cells exhibit only a high-threshold-activated (–30 to –20 mV) whole cell calcium channel current. When barium was used as the charge carrier, the high-threshold-activated current showed bi-exponential inactivation kinetics during a 500 ms voltage step from –90 to +10mV. The time constants of inactivation were approximately 75 and 750 ms. The fast inactivating component was more sensitive than the slow inactivating component to steady-state inactivation at depolarized holding potentials. The calcium channel current was inhibited by externally applied cadmium (10–300 M) and gadolinium (10–30 M) as well as by high concentrations of nickel and cobalt, Conus toxin (1 M) irreversibly blocked the calcium channel current. However, the dihydropyridine agonist, BAY K 8644 (3–10 M) and antagonists, nifedipine (3–10 M) and nimodipine (10 M) did not affect either component of the calcium channel current. Agents which blocked the calcium channel current did not exhibit any selectivity for the fast inactivating over the slow inactivating component of the current. These results indicate that whilst the calcium channel current recorded in differentiated SH-SY5Y cells can be classified on the basis of the blocking agents as being of the N type, the current shows more than one form of inactivation.     

Association between serum-soluble CD8 levels and parameters of immune activation in patients with human immunodeficiency virus infection

Summary We compared the serum concentrations of soluble CD8 with the immune activation markers neopterin, interferon-, tumour necrosis factor-, soluble CD4, and with CD34⁺ and CD38⁺ T-cell counts in patients with human immunodeficiency virus (HIV) infection. The majority of patients had increased concentrations of soluble CD8, interferon- and neopterin, and various significant correlations existed between them. Our results support the view that enhanced soluble CD8 levels indicate activated CD8⁺ T cells in patients with HIV infection.Abbreviations sCD8 serum-soluble CD8 - sCD4 serum-soluble CD4 - IFN- interferon- - TNF =tumour necrosis factor- - HIV human immunodeficiency virus - AIDS acquired immunodeficiency virus     

Untersuchungen zur Ultrastruktur lympho-epithelialer Thymustumoren unter besonderer Berücksichtigung der sog. „Emperipolesis“

Summary The ultrastructure of eleven thymomas with lymphocytic predominance, one epitheloid cell thymoma and two normal human thymuses is described with special reference to Emperipolesis. All patients have had myasthenia gravis.The normal human thymus consists of three parts: outer cortex, inner cortex, and medulla. The outer cortex contains mainly lymphoblasts and Metcalf's macrophages within the so-called Clark-packet's. The inner cortex consists mainly lymphocytes and interdigitating reticulum cells, and the medulla of epithelial cells, lymphocytes and Hassall's corpuscles.In all cases of lympho-epithelial thymoma and in normal human thymuses there are enormous interdigitations between epithelial (tumor) cells, lymphocytes and macrophages. The epitheloid cell thymomas also show findings which suggest an epithelial cell interaction. We have not found intact lymphocytes inside the cytoplasm of normal and/or tumor epithelial cells, macrophages or interdigitating reticulum cells.The intracellular existence of intact lymphocytes has been termed Emperipolesis by Humble, Jayne, and Pulvertaft, meaning internal wandering. These investigations indicate that Emperipolesis is not an adequate term for cellular interaction in normal human thymuses and thymomas. A false impression of intraepithelial location of thymic lymphocytes is created by two-dimensional sections of complex thymic structure. These ultrastructural studies revealed damage to lymphocytes only in macrophages with lymphocytolysis within these cells and accumulation of numerous heterophagic vacuoles containing fragments of lymphocytic debris within them.

FrÄulein C. Schürmann danke ich für die gute technische Assistenz, Herrn Priv.-Doz. Dr. med. R. W. Ch. Janzen, Neurologische Klinik der UniversitÄt Hamburg, für die klinischen Daten der Myasthenie-Patienten     

Expression and ligand-binding function of the integrin α4β1 (VLA-4) on neural-crest-derived tumor cell lines

Human neural-crest-derived tumor cell lines, including three neuroblastomas, an astrocytoma, a glioblastoma, a rhabdomyosarcoma and a melanoma were screened for the expression of the integrin ₄₁ (VLA-4). The neuroblastomas IMR-32 and SK-N-SH, the astrocytoma 131-INI, the glioblastoma Fogerty and the rhabdomyosarcoma TE-671 expressed ₄₁ as determined by cytofluorometry and immunoprecipitation. Another neuroblastoma line, LA-N-1, did not express ₄₁. Analysis of immunoprecipitated ₄₁ showed that the ₄ subunit from the various cell types differed in relative molecular weight (M _r ). The variability in the observed M _r could be accounted for by differences in the levels of N-linked glycosylation. The observed variability in M _r did not appear to affect function since intact cells and solubilized ₄₁ bound to a synthetic peptide identical in sequence to the CS-1 region of the alternatively spliced IIICS domain of fibronectin, a known ₄₁, ligand.     

Action of prostaglandin F2α on the course of pregnancy and plasma 17β-estradiol concentration in mice

The effect of prostaglandin F₂ (2 mg/kg at each stage of development) on the preimplantation development of mice and on their plasma 17-estradiol concentration was investigated. Prostaglandin F₂ inhibited mitotic division in the embryo and reduced the percentage of embryos shedding the zona pellucida. Meanwhile the 17-estradiol concentration in the peripheral blood plasma fell. Under physiological conditions there was a significant increase in the 17-estradiol concentration at the blastocyst stage after shedding of the zona pellucida.Department of Histology and Embryology, Pediatric Faculty, N. I. Pirogov Second Moscow Medical Institute. Laboratory of Endocrinology, Moscow University. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 88, No. 10, pp. 468–470, October, 1979.     

Acute ethanol consumption synergizes with trauma to increase monocyte tumor necrosis factor α production late postinjury

The hypothesis that acute ethanol uptake plus trauma can synergize to increase immunosuppression was tested. We found that, unlike non-alcohol-exposed patients, patients with acute alcohol use prior to trauma have a transient decrease in monocyte tumor necrosis factor (TNF) production during the very early postinjury (0–3 days) period. However, TNF production by these alcoholexposed patients' monocytes (MØ) became hyperelevated late postinjury (>9 days). Consequently, these massively elevated MØ TNF levels can contribute to posttrauma immunosuppression after acute alcohol use. We also demonstrate that normal monocyte activation with the superantigen,Staphylococcus enterotoxin B (SEB), results in a preferential induction of cellassociated MØ TNF production, described as characteristic of immunosuppressed trauma patients. Acutein vitro ethanol treatment down-regulated the elevated TNF production by trauma patients' MØ after either SEB, muramyl-dipeptide (MDP), interferon- plus MDP, or lipopolysaccharide (LPS) stimulation. Both SEB- and LPS-induced TNF mRNA induction was inhibited by acute alcohol treatment in normal MØ, indicating that ethanol can regulate cytokine gene expression. An additional immunosuppressive effect of acute ethanol's stimulation was suggested by its induction of elevated transforming growth factor production in trauma patients' activated MØ.     

Behavior of soluble HLA class I antigens in patients with chronic hepatitis C during interferon therapy: An early predictor marker of response?

Soluble HLA class I antigens (sHLA-I), ₂-microglobulin ( ₂-.) and alanine aminotransferase (ALT) serum levels have been evaluated in 16 patients affected by chronic hepatitis C treated for six months with recombinant interferon- (rIFN-, 3 MU three times a week). The predictor role of sHLA-I and ALT modifications with respect to the response to rIFN- therapy was also evaluated. Six patients responded (group 1), five patients relapsed following an initial response (group 2), and five did not respond to rIFN- treatment (group 3). The baseline serum levels of sHLA-I and ₂- were significantly higher in all three groups of HCV-positive patients with respect to HCV-negative controls (P<0.05). A significant increase of sHLA-I serum level with respect to baseline value (P<0.001) was observed in group 1 patients after two weeks of rIFN- treatment. sHLA-I serum level then decreased, although remaining steadily and significantly increased with respect to baseline (P values ranging from 0.05 to 0.01) in the following five months and then returned to baseline one month after the end of rIFN- administration. No significant variations of ₂- serum levels were detected throughout the observation period. In group 1 patients ALT serum levels significantly decreased after two weeks of rIFN- treatment (P<0.001) and then remained in the normal range throughout the observation period. In the other two groups of patients no relevant variations of sHLA-I and ₂- serum levels were found during and after rIFN- therapy. The modifications of sHLA-I serum levels discriminate, as a single marker, group 1 patients from group 2 and 3 patients after two weeks of rIFN- treatment (P<0.003). The association of sHLA-I and ALT modifications improves the discriminant power and leads to a complete differentiation of the three groups of patients after four weeks of rIFN- treatment (P<0.0001). If confirmed in a larger series of patients, these results will provide a useful marker to predict which patients affected by chronic hepatitis C will respond to treatment and will help to avoid their ineffective treatment with an expensive and potentially harmful drug.     

Molecular localization of human IgG anti-F(ab′)2 reactivity with variable- and constant-region λ light-chain epitopes

Human IgG antibodies reacting with antigenic determinants on F(ab)₂ fragments represent generic antiidiotypic antibodies present in the serum of normal individuals. Additionally, the titers of these antibodies in the sera of patients with systemic lupus erythematosus (SLE) are inversely related to disease activity. Because these autoantibodies recognize predominantly light chain-related epitopes, especially type, we synthesized constant (C)- and variable (V)-related overlapping 7-mer peptides on polypropylene pins to determine anti-F(ab)₂-reactive epitopes on human light chains. ELISA reactivity of affinity-purified anti-F(ab)₂ antibodies obtained from normal individuals and from patients with SLE, as well as murine anti-human light-chain monoclonal antibodies specific for C and V subgroup-related determinants, was tested using the overlapping 7-mers of human light-chain sequence. The patterns of reactivity against C-related peptides were similar in both normal and SLE-derived anti-F(ab)₂ antibodies. However, reactivity profiles against V-related peptides were distinctively different between the normal and the SLE-associated anti-F(ab)₂ autoantibodies. A decrease in reactivity among the SLE IgG anti-F(ab)₂ antibodies was noted for particular amino acid V complementarity-determining region (CDR) residues, including glycine at positions 27 and 54, alanine at 16 and 37, and tyrosine at 28 and 91. This different pattern of reactivity from normal may indicate that in SLE there is a failure of antiidiotypic control mechanisms, as reflected by a defect in production of antibody to immunodominant V CDR residues.     

Resistance toTrypanosoma cruzi infection in relation to the timing of IgG humoral response

The Biozzi high (BH) and low (BL) responder mice (Selection III) differeed in their susceptibility toTrypanosoma cruzi. The BH strain responded quickly to the infection, similar to the reaction of (CBA×C57B1/10)F₁ mice but in contrast to the susceptible BL strain. We suggest that the IgG response mounted by the host during the prepatent period of the infection is crucial to the outcome of the infection.     

Two types of potassium channel regulated by ATP in pancreatic B cells isolated from a type-2 diabetic human

Two types of K channel regulated by ATP were observed in pancreatic cells from a type-2 diabetic man. One type had a conductance of 67 pS at-70 mV in symmetrical 140 mM KCl and was inhibited by intracellular ATP with a half-maximal concentration of 40 M. ATP inhibition was antagonised by ADP. Tolbutamide inhibited the whole-cell K currents half-maximally at 25 M. This channel has properties similar to those found for the ATP-sensitive K channel in rodent and normal human cells. The second channel type observed was an ATP-activated K channel. It had a conductance of 37 pS at -70 mV in symmetrical 140 mM KCl and was activated half-maximally by 9 M intracellular ATP. This channel was unaffected by 1 mM tolbutamide. In cell-attached patches, one cell out of four tested responded to 20 mM glucose with depolarization. The role of the ATP-activated K channel with respect to the (patho)physiology of the cell is uncertain.     

Secretory characteristics of pancreatic γ-glutamyl transpeptidase

Summary In an attempt to identify the secretory mechanism of pancreatic -glutamyl transpeptidase (-GTP), constant intravenous infusions of secretin alone and in combination with caerulein were performed in anesthetized dogs prepared with a pancreatic fistula. Caerulein produced a marked increase in amylase concentration and only a slight increase in -GTP. -GTP concentration of the pancreatic juice varied from 12 to 490 mU per ml which ranged up to 188-fold higher than that of the serum. The enzyme concentration depended largely on the flow rate, revealing 3 characteristic curvlinear relationship, regardless of whether caerulein was added to the secretin infusion. No significant relation was demonstrated between amylase concentration and flow rate, amylase and -GTP concentrations, and -GTP and protein concentration. An inverse linear correlation between -GTP and chloride concentrations was obtained when flow rate was below 2.5 ml per 15 min. A significant linear relationship was demonstrated between -GTP and leucine aminopeptidase concentrations, and amylase and protein concentrations. The results presented clearly demonstrate that the mechanism of pancreatic secretion of -GTP is quite distinct from that of amylase.Supported in part by Cancer Research Grant from the Welfare Ministry of Japan.     

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to –60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 M) and amiloride (500 M) and was insensitive to -conotoxin (-CgTx) (4 M) and to the Ca agonist Bay K 8644 (5 M). The high-voltage-activated (HVA) Ca current turned on positive to –40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. CgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the -CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) -CgTx-sensitive and Bay-K-8644-insensitive (-S/BK-I) and (ii) -CgTx-insensitive and Bay-K-8644-sensitive (-I/BK-S), the latter displaying a stronger Cadependent inactivation.