Cryptosporidium oocysts in Bent mussels (Ischadium recurvum) in the Chesapeake Bay

Filter-feeding molluscan shellfish can concentrate environmentally derived waterborne pathogens of humans, which can be utilized in the sanitary assessment of water quality. In the present study, oocysts of Cryptosporidium were detected in Bent mussels (Ischadium recurvum) at two Chesapeake Bay sites from which C. parvum-contaminated oysters had previously been collected. Spiking of Cryptosporidium-free blue mussel (Mytilus edulis) tissue with C. parvum oocysts showed a 51.1% recovery rate of oocysts, giving an oocyst detection limit of 19 oocysts/0.7 ml of mussel tissue homogenate. The results indicate that Bent mussels, which are common throughout the Chesapeake Bay region, may prove to be useful as biological indicators of water contamination with Cryptosporidium oocysts. Received: 24 November 1998 / Accepted: 20 January 1999     

Pulmonary blood volume and its effects on pressure/flow relations and flow resistance in isolated lungs of rabbits

 Quantitative information about the effects of pulmonary blood volume (Q _p) on pulmonary haemodynamics is lacking since Q _p changes inevitably with flow. To separate flow-dependent from volume-dependent changes in intravascular pressures we imposed changes in Q _p (measured continuously) by altering outflow pressure in seven isolated, blood-perfused rabbit lungs and studied the effects of Q _p on the relations between arteriovenous pressure gradient (ΔP) and blood flow (Q·) under two conditions: flow-dependent volume changes were either permitted or compensated. In the latter circumstances, ΔP changed more for a given change in Q·. The ΔP/Q· relations were shifted to smaller ΔP when Q _p was increased. Hence, the calculated flow resistance (R = ΔP/Q·) decreased with increasing Q _p at a given Q·. Assuming constant viscosity, changes in R can be predicted from changes in vessel geometry and thus Q _p. We found that R increased less than expected (by a factor of 3–7.5 instead of 9) when Q _p was reduced to one-third. This discrepancy may be explained by a change in blood distribution within the lung despite constant Q _p and by a change in apparent blood viscosity with Q·. Regardless of these speculations we have shown that Q _p determines ΔP at each flow and thus flow resistance. Received: 21 March 1997 / Received after revision and accepted: 3 September 1997     

A comparative scale of autonomic function with age through the tone-entropy analysis on heart period variation

Tremendous numbers of heart rate variability studies have aimed to elucidate age-associated alterations of autonomic function in the past decades. However, the studies, far from clarifying ageing mechanisms, fell into confusion by a lack of common scales. The purpose of this study is to show a possibility to establish a comparative scale of autonomic function through a method, tone-entropy (T-E) analysis on heart period variation, whose validity has been already examined on typical physiological cases (Oida et al. in J Appl Physiol 82:1794–1801, 1997; Oida et al. in J Gerontol 54A:M219–M224, 1999a; Oida et al. in Acta Physiol Scand 165:129–134, 1999b; Oida et al. in Acta Physiol Scand 165:421–422, 1999c; Amano et al. in Eur J Appl Physiol 94:602–610, 2005). In this study, 276 subjects from teens to seventies were examined at rest by T-E analysis together with conventional time and frequency domain analyses. The tone (negativity represents vagal predominance) became significantly high [−0.174 ± 0.026 (teens) to −0.024 ± 0.004 (seventies), P < 0.05 for one-way ANOVA], and the entropy (total autonomic activity), significantly low [4.40 ± 0.12 (teens) to 2.90 ± 0.09 bit (seventies), P < 0.05] with advancing age. The result, plotted in 2-D T-E space, showed that the ageing traced a curvi-linear relation from right-bottom to left-top, and was consistent with previously studied typical physiological cases. The conventional analyses showed almost the same autonomic reduction as T-E did, but failed in detecting delicate alteration of autonomic balance. The results, showing that autonomic activity reduced in both pathways impairing vagal predominance significantly with ageing, suggested a possibility to assess autonomic function in 2-D T-E space in a comparative way.     

<Emphasis Type="Italic">In Vivo</Emphasis> Dynamic Deformation of the Mitral Valve Annulus

Though mitral valve (MV) repair surgical procedures have increased in the United States [Gammie, J. S., et al. Ann. Thorac. Surg. 87(5):1431–1437, 2009; Nowicki, E. R., et al. Am. Heart J. 145(6):1058–1062, 2003], studies suggest that altering MV stress states may have an effect on tissue homeostasis, which could impact the long-term outcome [Accola, K. D., et al. Ann. Thorac. Surg. 79(4):1276–1283, 2005; Fasol, R., et al. Ann. Thorac. Surg. 77(6):1985–1988, 2004; Flameng, W., P. Herijgers, and K. Bogaerts. Circulation 107(12):1609–1613, 2003; Gillinov, A. M., et al. Ann. Thorac. Surg. 69(3):717–721, 2000]. Improved computational modeling that incorporates structural and geometrical data as well as cellular components has the potential to predict such changes; however, the absence of important boundary condition information limits current efforts. In this study, novel high definition in vivo annular kinematic data collected from surgically implanted sonocrystals in sheep was fit to a contiguous 3D spline based on quintic-order hermite shape functions with C² continuity. From the interpolated displacements, the annular axial strain and strain rate, bending, and twist along the entire annulus were calculated over the cardiac cycle. Axial strain was shown to be regionally and temporally variant with minimum and maximum values of −10 and 4%, respectively, observed. Similarly, regionally and temporally variant strain rate values, up to 100%/s contraction and 120%/s elongation, were observed. Both annular bend and twist data showed little deviation from unity with limited regional variations, indicating that most of the energy for deformation was associated with annular axial strain. The regionally and temporally variant strain/strain rate behavior of the annulus are related to the varied fibrous-muscle structure and contractile behavior of the annulus and surrounding ventricular structures, although specific details are still unavailable. With the high resolution shape and displacement information described in this work, high fidelity boundary conditions can be prescribed in future MV finite element models, leading to new insights into MV function and strategies for repair. Chad Eckert and Brett Zubiate contributed equally to this work.     

Electron microscope observations on Onchocerca ochengi and O. fasciata (Nematoda: Filarioidea)

The fine structure of the females and males of Onchocerca ochengi (parasitizing zebu and cattle) and of the females of O. fasciata from camels were described and compared to other filariae of the genus Onchocerca. It was shown that O. ochengi resembles O. volvulus of humans in its degree of development, while being more primitive than O. gibsoni. Besides other similarities O. ochengi attracts inflammatory cells in the way of O. volvulus and these could be a model for chemotherapeutic trials. Received: 15 January 1997 / Accepted: 1 February 1997     

Trypanosoma cruzi in the rectum of the bug Triatoma infestans : effects of blood ingestion of the vector and artificial diuresis

The population density and the percentage of different developmental stages of an established infection of Trypanosoma cruzi were determined at 40 days after the last feeding of the fourth instar in the rectum (lumen, anterior and posterior wall) of fed and unfed groups of fifth instars of Triatoma infestans. Additionally, the rectum and the Malpighian tubules were incubated in saline, inducing diuresis by addition of the diuretic hormone. The rectum contained an average of 200,000–400,000 T. cruzi. After feeding the percentages of spheromastigotes and drop-like intermediate stages were reduced from <7% and 15%, respectively, to <3%, but those of slender intermediate stages increased statistically significantly from <7% to 10%. After 4 h of diuresis the in-vitro-incubated isolated rectum with the four Malpighian tubules showed the same trends, indicating that diuresis rather than factors of the hemolymph or digestive products induces the development of metacyclic trypomastigotes of T. cruzi originating from epimastigotes. Received: 6 April 1997 / Accepted: 7 May 1997     

Candida albicans ALS3 and insights into the nature of the ALS gene family

The ALS1 (agglutinin-like sequence) gene of Candida albicans encodes a protein similar to alpha-agglutinin, a cell-surface adhesion glycoprotein of Saccharomyces cerevisiae (Hoyer et al. 1995). A central domain of a tandemly repeated 108-bp sequence is found in the ALS1 coding region. This tandem-repeat motif hybridizes to multiple C. albicans genomic DNA fragments, indicating the possibility of other ALS1-like genes in C. albicans (Hoyer et al. 1995). To determine if these fragments constitute a gene family, tandem-repeat-hybridizing genomic fragments were isolated from a fosmid library by PCR screening using primers based on the consensus tandem-repeat sequence of ALS1 (Hoyer et al. 1995). One group of fosmids, designated ALS3, encodes a gene with 81% identity to ALS1. The sequences of ALS1 and ALS3 are most conserved in the tandem-repeat domain and in the region 5′ of the tandem repeats. Northern-blot analysis using unique probes from the 3′ end of each gene demonstrated that ALS1 expression varies, depending on which C. albicans strain is examined, and that ALS3 is hyphal-specific. Both genes are found in a variety of C. albicans and C. stellatoidea strains examined. The predicted Als1p and Als3p exhibit features suggesting that both are cell-surface glycoproteins. Southern blots probed with conserved sequences from the region 5′ of the tandem repeats suggest that other ALS-like sequences are present in the C. albicans genome and that the ALS family may be larger than originally estimated. Received: 24 December 1997 / 12 March 1998     

Permissiveness of two African wild rodents, Mastomys huberti and Arvicanthis niloticus, to Schistosoma intercalatum: epidemiological consequences

The compatibility between Schistosoma intercalatum (Cameroon) and two wild rodents commonly found in Africa, Mastomys huberti (the multimammate mouse) and Arvicanthis niloticus (the Nile rat) was studied to determine their biological capacities to act as hosts for S. intercalatum. In both rodent species the general mean worm recovery was high (33 ± 0.1% in M. huberti and 33.8 ± 0.1% in A. niloticus) and worm mortality was very low from 6 to 20 weeks postinfection; parasite maturity was reached. The high number of released eggs as well as the viability and the infectivity of the miracidia to the snail vector showed that M. huberti and A. niloticus are very permissive to S. intercalatum and may act as hosts for the human disease. Received: 19 November 1996 / Accepted: 15 January 1997     

Glomerular volume and the glomerular vascular pole area in patients with insulin-dependent diabetes mellitus

 The vascular pole area (VPA) and glomerular volume were measured in renal biopsies from 9 insulin-dependent diabetes mellitus (IDDM) patients with normal albumin excretion rate (IDDM group 1), 38 IDDM patients with albumin excretion rate >15 μg/min (IDDM group 2) and 10 living kidney donors (ND). The volume of individual glomeruli was estimated as the sum of profile areas factored by the measured distance between levels, t∼ 10 μm, and VPA as the sum of chords multiplied by t. Mean glomerular volume was increased in IDDM patients but reached statistical significance only in IDDM group 2 (P = 0.002 vs ND). VPA was significantly different among the groups, mean (CV%) was 2036 (29) μm² in ND, 3555 (34) μm² in IDDM group 1, and 3528 (48) μm² in IDDM group 2, p = 0.004 and 0.001, IDDM versus ND. VPA calculated as a percentage of the surface area of the corresponding glomerulus was 2.4 (23)% in ND, 3.4 (27)% in IDDM group 1, and 3.3 (42)% in IDDM group 2; P = 0.007 and 0.01, IDDM versus ND. The intra-biopsy coefficient of variation was high (20–35%) and of the same order in all groups for all three measurements. Glomerular volume and absolute as well as relative size of VPA showed a positive correlation with estimates of mesangial expansion in IDDM group 2 and the VPA showed a negative correlation with GFR. Thus, part of the enlargement may represent a compensatory phenomenon triggered by the development of structural and functional abnormalities in the diabetic kidney. Received: 22 April 1997 / Accepted: 20 June 1997     

The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl– conductance of Xenopus oocytes

 Oocytes from Xenopus laevis activate a Ca²⁺ dependent Cl^– conductance when exposed to the Ca²⁺ ionophore ionomycin. This Ca²⁺ activated Cl^– conductance (CaCC) is strongly outwardly rectifying and has a halide conductivity ratio (G_I– / G_Cl–) of about 4.4. This is in contrast to the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl^– conductance, which produces more linear I/V curves with a G_I– / G_Cl– ratio of about 0.52. Ionomycin enhanced CaCC (ΔG) in water injected and CFTR expressing ooyctes in the absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/l) by (μS) 23 ± 1.9 (n=9) and 23.6 ± 2.3 (n=11). Stimulation by IBMX did not change CaCC in water injected oocytes. CaCC was inhibited in CFTR-expressing ooyctes after stimulation with IBMX or a membrane permeable form of cAMP and was only 5.1 ± 0.48 μS (n=18) and 6.9 ± 0.6 (n=3), respectively. Inhibition of CaCC was correlated to the amount of CFTR-current activated by IBMX. ΔF508-CFTR which demonstrates only a small residual function in activating a cAMP dependent Cl^– channel in oocytes inhibited CaCC to a lesser degree (ΔG=12.1 ± 1.1 μS; n=7). Changes of CFTR and CaCC-Cl^– whole cell conductances were also measured when extracellular Cl^– was replaced by I^–. The results confirmed the reduced activation of CaCC in the presence of activated CFTR. No evidence was found for inhibition of CFTR-currents by increase of intracellular Ca²⁺. Moreover, intracellular cAMP was not changed by ionomycin and stimulation by IBMX did not change the ionomycin induced Ca²⁺ increase in Xenopus oocytes. Taken together, these results suggest that activation of CFTR-Cl^– currents is paralleled by an inhibition of Ca²⁺ activated Cl^– currents in ooyctes of Xenopus laevis. These results provide another example for CFTR-dependent regulation of membrane conductances other than cAMP-dependent Cl^– conductance. They might explain previous findings in epithelial tissues of CF-knockout mice. Received: 17 June 1997 / Received after revision: 4 September 1997 / Accepted: 5 September 1997     

Inhibition by inositoltetrakisphosphates of calcium- and volume-activated Cl– currents in macrovascular endothelial cells

 We have used the whole-cell patch-clamp technique to study the effects of inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P ₄], inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P ₄] and inositol 1,3,4,5,6-pentacisphosphate [Ins(1,3,4,5,6)P ₅] on volume-activated Cl^– currents (I _Cl,vol) in cultured endothelial cells from bovine pulmonary artery (CPAE cells). Ins(1,4,5,6)P ₄ and Ins(3,4,5,6)P ₄ were applied intracellularly via the patch pipette at concentrations between 10 and 100 μM. Both tetrakisphosphates inhibited the Cl^– current I _Cl,Ca, which was activated by intracellular loading of the cells with 500 nM Ca²⁺ [for inhibition by Ins(1,4,5,6)P ₄: 58% at 10 μM, 75% at 100 μM; for Ins(3,4,5,6)P ₄: 44% at 10 μM, 65% at 100 μM]. Inhibition of I _Cl,Ca occurred without significant changes in its kinetic properties. The amplitude of I _Cl,vol activated by a 13.5 or 27% hypotonic solution at +100 mV was strongly reduced in cells loaded with either tetrakisphosphate, i.e. a 73% reduction for Ins(3,4,5,6)P ₄ and 89% for Ins(1,4,5,6)P ₄ at 100 μM. Both tetrakisphosphates also inhibited a current probably identical to I _Cl,vol which was activated by dialysing the cell with 100 μM guanosine 5′-O-(3-thiotriphosphate) (GTP[γ-S]). Ins(1,3,4,5,6)P ₅ at a concentration of 30 μM did not significantly reduce I _{Cl, vol}. The effects of Ins(3,4,5,6)P ₄ may represent an inhibitory pathway for the I _Cl,Ca and I _Cl,vol in macrovascular endothelium after sustained receptor-mediated activation of phospholipase C. Received: 8 September 1997 / Received after revision and accepted: 31 October 1997     

Connecting filament mechanics in the relaxed sarcomere

By examining the mechanical properties of single unactivated myofibrils it has been shown that shortening and stretching of sarcomeres occurs in stepwise fashion, and that steps are seen also in the relaxed state (Yang et al. (1998) Biophys J 74: 1473–1483; Blyakhman et al. (2001) Biophys J 81: 1093–1100; Nagornyak et al. (2004) J. Muscle. Res. Cell Motil. 25: 37–43). The latter are inevitably associated with connecting filaments. Here, we carried out measurements on single myofibrils from rabbit psoas muscle to investigate steps in unactivated specimens in more detail. Myofibrils were stretched and released in ramp-like fashion. For the single sarcomere the time course of length change was consistently stepwise. We found that in the unactivated myofibrils, step size depended on initial sarcomere length, diminishing progressively with increase of initial sarcomere length, whereas in the case of activated sarcomeres, step size was consistently 2.7 nm.     

Interference of a short-chain phospholipid with ion transport pathways in frog skin

 The effects of mucosal application of the short-chain phospholipid didecanoyl-L-α-phosphatidylcholine (DDPC; with two saturated 10-carbon acyl chains) on active Na⁺ transport and transepithelial conductance (G) in the frog skin (Rana temporaria) were investigated. Active Na⁺ transport was measured as the amiloride-sensitive short-circuit current (I _SC) and G was determined from transepithelial voltage-clamp pulses under short-circuit conditions. DDPC dose-dependently inhibited I _SC with an ID₅₀ of about 0.05% (w/v) and a maximal effect (≈55%) at ≥ 1% DDPC. G increased to steady-state values above control level. Simultaneously, equal increases in unidirectional sucrose permeabilities (P _Su; measured from [¹⁴C]sucrose fluxes) were observed, and a positive correlation was demonstrated between DDPC-induced changes in P _Su and G. Since amiloride did not prevent the increase in G by DDPC, these results suggest that the DDPC-induced increase in G represents an increase in the paracellular shunt conductance. The effects of mucosal DDPC were almost fully reversible within 8 h. The results indicate that DDPC inhibits amiloride-sensitive Na⁺ channels in the apical membrane of the frog skin epithelium and opens a paracellular tight junction pathway. Both effects may be caused by incorporation of DDPC in the apical cell membrane. Received: 28 November 1996 / Received after revision: 17 March 1997 / Accepted: 21 March 1997     

Surface charge and surface carbohydrates of cryptobia salmositica virulent and avirulent forms and of C. bullocki (Kinetoplastida: Cryptobiidae)

The surface charge and surface carbohydrate residues of the virulent (freshly isolated from the fish blood) and avirulent forms (from culture) of Cryptobia salmositica and one strain of C. bullocki were studied. Measurements of the zeta potential of parasites showed that C. bullocki and the virulent form of C. salmositica had a net negative surface charge of about −15 mV, whereas the attenuated form of C. salmositica showed a surface charge of −7.9 mV. Enzymatic treatments of parasites with neuraminidase, trypsin, or phospholipase C indicated the presence of sialic acid residues, phosphate groups, and protein glycoconjugates as components of the Cryptobia surface that accounted for their surface charge. Residues of α-D-man, α- and β-D-gal, α-D-galNAc, α-L-fuc, and D-glcNAc could be detected on the surface of all parasites by specific fluorescein isothiocyanate (FITC)- and colloidal gold-labeled lectins. The cell surface of the avirulent form of C. salmositica showed the strongest reactivity to almost all lectins tested. A remarkable binding pattern of lectins in the anterior region of parasites was observed. Received: 1 October 1996 / Accepted: 24 February 1997     

Reciprocity between membranous and nuclear expression of β-catenin in colorectal tumours

 β-Catenin has a central role not only in linking the cadherin-mediated cell adhesion system but also in the intercellular signalling pathway. To investigate alterations of β-catenin in the development of colorectal carcinoma, the pattern of β-catenin expression was studied using immunohistochemistry in 74 sporadic colorectal adenomas, in histologically normal mucosa adjacent to 65 of these adenomas, and in 52 carcinomas arising in adenomas. All normal epithelia displayed cell boundary staining for β-catenin. Adenomas and carcinomas showed varying degrees of membranous staining. However, some tumours also showed nuclear staining of β-catenin protein. Decreased membranous and increased nuclear β-catenin staining were associated with increasing degrees of dysplasia in adenomas (P < 0.005, P < 0.05, respectively). Carcinomas manifested significantly reduced membranous, but enhanced nuclear β-catenin expression compared with their associated adenomas (P < 0.001, P < 0.005, respectively). An inverse correlation was found between decreased membranous and increased nuclear staining of β-catenin in both adenomas and carcinomas (P < 0.025, P < 0.05, respectively). The data confirm that reduced membranous and increased nuclear expression of β-catenin is associated with the progression of colorectal adenomas to carcinomas. Our results also suggest that decreased membranous expression of β-catenin may result from aberrant localisation of the protein in the cell nucleus. Received: 1 January 1997 / Accepted: 26 February 1997     

In vitro phagocytosis of Giardia duodenalis cysts by hemocytes of the Asian freshwater clam Corbicula fluminea

Hemocytes of the Asian freshwater clam Corbicula fluminea, phagocytosed in vitro infectious Giardia duodenalis cysts. After 15, 30, 60, 90, and 120 min of incubation an average of 22%, 32%, 43%, 54%, and 72% of the cysts were phagocytosed by 22%, 55%, 63%, 81%, and 86% of the hemocytes, respectively. The number of hemocytes showing phagocytosis and the mean number of cysts ingested per hemocyte increased␣significantly over time (P < 0.01); the numbers of nonphagocytosed cysts significantly decreased (P < 0.02). Extrapolation reveals that C. fluminea can retain by phagocytosis an average of 1.6 × 10⁶ G. duodenalis cysts/ml hemolymph. The phagocytic capacity of C. fluminea hemocytes indicates the applicability of this freshwater benthic bivalve for bioindication of contamination of waste waters and agricultural drainage with Giardia cysts. Received: 28 March 1997 / Accepted: 6 June 1997     

Babesia bovis : identification of immunodominant merozoite surface proteins in soluble culture-derived exoantigen

Babesia bovis merozoite proteins presenting as exoantigens in in vitro culture supernatants have been characterized. Bovine antisera to B. bovis exoantigens were used to immunoprecipitate [³⁵S]-methionine metabolically labeled or lactoperoxidase-catalyzed radioiodinated B. bovis merozoite proteins. A total of 24 metabolically labeled proteins ranging in molecular weight from 24,000 to 225,000 Da and 9 radioiodinated proteins with molecular weights varying between 24,000 and 225,000 Da were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Monoclonal antibodies to B. bovis merozoite surface proteins were also used to immunoprecipitate metabolically labeled exoantigens directly from in vitro culture supernatants. These results demonstrate epitopes from at least nine merozoite surface proteins present in the exoantigen fraction, among which are the recently characterized major surface antigens 1 and 2, rhoptry-associated protein 1, and spherical body protein 2. Received: 1 April 1997 / Accepted: 6 May 1997     

Sequence and immunogenicity of the Taenia saginata homologue of the major surface antigen of Echinococcus spp.

A clone (R-Tso18) was isolated from a Taenia saginata oncosphere cDNA library by screening with sera from rabbits immunised with oncosphere extract. It contained a full-length cDNA sequence of 1893 bp with an open reading frame of 1680 bp, corresponding to 559 amino acids with a deduced molecular mass of 65.173 kDa and an isoelectric point of 6.08. The R-Tso18 protein showed 80–84% nucleotide identity with the major protoscolex surface antigens of Echinococcus multilocularis (EM10) and E. granulosus (EG10). Preliminary immunogenicity studies employing the radio-labeled R-Tso18 protein in immune co-precipitation assays indicated sero-positivity for T. saginata-infected calf sera (6/13), T. solium cysticercosis human (7/22) and pig (2/2) sera and E. multilocularis (6/10)- and E. granulosus (1/12)-infected human sera, whereas other helminth-infection sera were negative. As immuno-precipitation is a relatively insensitive assay, it was concluded that further studies on the diagnostic potential of the purified recombinant R-Tso18 antigen, or its peptides, are merited. Received: 9 July 1997 / Accepted: 3 November 1997     

On a branching model of division-within-division

We consider a deterministic version of a stochastic model fordivision-within-division processes described by Kimmel (1997,in: Proceedings of the IMA Workshop ‘Classical and ModernBranching Processes’, K. Athreya and P. Jagers, eds.,New York, Springer). It is shown that the behaviour of the deterministicmodel can be analysed by using an associated Markov chain, usingthe methods of Barbour et al. (1996, Ann. Appl. Prob. 6, 1045–1074).     

Cholinergic agonists decrease quantal output at the frog neuromuscular junction by targeting a calcium channel blocked by ω-conotoxin

 Nicotinic cholinergic agonists are known to decrease synchronous evoked quantal output at the frog neuromuscular junction [Van der Kloot 1993, J Physiol (Lond) 468:567–589]. Here we also show that carbachol decreases the frequency of miniature endplate potentials (F _MEPP) in solutions containing elevated levels of K⁺ and Ca²⁺. Carbachol did not decrease F _MEPP in hypertonic solutions or in solutions containing the Ca²⁺ ionophore ionomycin and Ca²⁺. We conclude that the nicotinic agonists decrease Ca²⁺ influx through voltage-gated Ca²⁺ channels. Carbachol did not alter two-pulse facilitation. A blocker of N-type Ca²⁺ channels, ω-conotoxin GVIA, antagonized the nicotinic agonist-induced decrease in evoked quantal output. The effect of carbachol was not altered by ω-conotoxin MVIIC, a blocker of P-type and certain other Ca²⁺channels. The Ca²⁺ channel targeted by the nicotinic agonists appears to be of the N-type. Received: 6 March 1997 / Received after revision: 6 May 1997 / Accepted: 4 June 1997