Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers.

A major group of natural killer (NK) T cells express an invariant Valpha14(+) T cell receptor (TCR) specific for the lipoglycan alpha-galactosylceramide (alpha-GalCer), which is presented by CD1d. These cells may have an important immune regulatory function, but an understanding of their biology has been hampered by the lack of suitable reagents for tracking them in vivo. Here we show that tetramers of mouse CD1d loaded with alpha-GalCer are a sensitive and highly specific reagent for identifying Valpha14(+) NK T cells. Using these tetramers, we find that alpha-GalCer-specific T lymphocytes are more widely distributed than was previously appreciated, with populations of largely NK1.1(-) but tetramer-binding T cells present in the lymph nodes and the intestine. Injection of alpha-GalCer leads to the production of both interferon gamma and interleukin 4 by nearly all NK T cells in the liver and the majority of the spleen within 2 h. These cells mostly disappear by 5 h, and they do not reappear after 1 wk. Curiously, tetramer-positive thymocytes do not rapidly synthesize cytokines, nor do they undergo decreases in cell number after lipid antigen stimulation, although they express equivalent TCR levels. In summary, the data presented here demonstrate that alpha-GalCer-specific NK T cells undergo a unique and highly compartmentalized response to antigenic stimulation.

Ras mediates effector pathways responsible for pre-B cell survival, which is essential for the developmental progression to the late pre-B cell stage.

Ras is essential for the transition from early B cell precursors to the pro-B stage, and is considered to be involved in the signal cascade mediated by pre-B cell antigen receptors. To examine the role of p21(ras) in the late stage of B cell differentiation, we established transgenic mice (TG) expressing a dominant-inhibitory mutant of Ha-ras (Asn-17 Ha-ras) in B lineage cells at high levels after the early B cell precursor stage. Expression of p21(Asn-17) (Ha-ras) was associated with a prominent reduction in the number of late pre-B cells, but had little effect on proliferation of early pre-B cells. Inhibition of p21(ras) activity markedly reduced the life span of pre-B cells, due, at least in part, to downregulation of the expression of an antiapoptotic protein, Bcl-xL. Thus, the apparent role for p21(ras) activity in pre-B cell survival may explain the decreased numbers of late pre-B cells in Asn-17 Ha-ras TG. Consistent with this possibility, overexpression of Bcl-2 in Asn-17 Ha-ras TG reversed the reduction in the number of late pre-B cells undergoing immunoglobulin light chain gene (IgL) rearrangement and progressing to immature B cells. These results suggest that p21(ras) mediates effector pathways responsible for pre-B cell survival, which is essential for progression to the late pre-B and immature B stages.

Noble gas partitioning between metal and silicate under high pressures.

Measurements of noble gas (helium, neon, argon, krypton, and xenon) partitioning between silicate melt and iron melt under pressures up to 100 kilobars indicate that the partition coefficients are much less than unity and that they decrease systematically with increasing pressure. The results suggest that the Earth's core contains only negligible amounts of noble gases if core separation took place under equilibrium conditions.

The nanostructure and hydrogenation reaction of Mg50Co50 BCC alloy prepared by ball-milling.

Mg50Co50 alloy before and after hydrogenation was investigated by means of transmission electron microscopy (TEM). Mg50Co50 alloy before hydrogenation was found to contain crystals not larger than 5 nm in size. Selected-area electron diffraction patterns (SAEDPs) revealed that these nanocrystals have a body-centered cubic (BCC) structure with a lattice parameter of about 0.3 nm. Distribution of Mg and Co elements in the Mg50Co50 alloy was uniform, indicated by energy dispersive x-ray spectroscopy (EDS) analysis. Crystallization and decomposition occurred in the Mg50Co50 alloy during hydrogenation. A large number of crystals larger than 10 nm were observed in the hydrogenated sample. The SAEDPs showed polycrystalline rings corresponding to the BCC phase and the Co metal phase. The existence of Mg-rich Mg-Co crystals and Co particles was also confirmed by TEM-EDS analysis.

Reversal of lidocaine effects on sodium currents by isoproterenol in rabbit hearts and heart cells.

We demonstrated recently that isoproterenol enhanced the cardiac voltage-dependent sodium currents (INa) in rabbit ventricular myocytes through dual G-protein regulatory pathways. In this study, we tested the hypothesis that isoproterenol reverses the sodium channel blocking effects of class I antiarrhythmic drugs through modulation of INa. The experiments were performed in rabbit ventricular myocytes using whole-cell patch-clamp techniques. Reversal of lidocaine suppression of INa by isoproterenol (1 microM) was significant at various concentrations of lidocaine (20, 65, and 100 microM, P < 0.05). The effects of isoproterenol were voltage dependent, showing reversal of INa suppression by lidocaine at normal and hyperpolarized potentials (negative to -80 mV) but not at depolarized potentials. Isoproterenol enhanced sodium channel availability but did not alter the steady state activation or inactivation of INa nor did it improve sodium channel recovery in the presence of lidocaine. The physiological significance of the single cell INa findings were corroborated by measurements of conduction velocities using an epicardial mapping system in isolated rabbit hearts. Lidocaine (10 microM) significantly suppressed epicardial impulse conduction in both longitudinal (theta L, 0.430 +/- 0.024 vs. 0.585 +/- 0.001 m/s at baseline, n = 7, P < 0.001) and transverse (theta T, 0.206 +/- 0.012 vs. 0.257 +/- 0.014 m/s at baseline, n = 8, P < 0.001) directions. Isoproterenol (0.05 microM) significantly reversed the lidocaine effects with theta L of 0.503 +/- 0.027 m/s and theta T of 0.234 +/- 0.015 m/s (P = 0.014 and 0.004 compared with the respective lidocaine measurements). These results suggest that enhancement of INa is an important mechanism by which isoproterenol reverses the effects of class I antiarrhythmic drugs.

Presentation of self and microbial lipids by CD1 molecules.

CD1 molecules present both self lipids and microbial lipids. Recent studies have elucidated novel antigenic structures that can be presented by CD1 for T cell stimulation, as well as new pathways for lipid-antigen presentation. Additionally, the development of lipid-CD1 tetramers now permits the tracking of CD1-reactive T cells during immune responses. Despite this, the roles of CD1-reactive T cells in both host defense and immune regulation remain to be unequivocally defined.

CLOCK is involved in obesity-induced disordered fibrinolysis in ob/ob mice by regulating PAI-1 gene expression.

BACKGROUND: An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. AIM: The present study investigates whether the circadian clock component CLOCK is involved in obesity-induced PAI-1 elevation. METHODS: We examined plasma PAI-1 and mRNA expression levels in tissues from leptin-deficient obese and diabetic ob/ob mice lacking functional CLOCK protein. RESULTS: Our results demonstrated that plasma PAI-1 levels were augmented in a circadian manner in accordance with the mRNA expression levels in ob/ob mice. Surprisingly, a Clock mutation normalized the plasma PAI-1 concentrations in accordance with the mRNA levels in the heart, lung and liver of ob/ob mice, but significantly increased PAI-1 mRNA levels in adipose tissue by inducing adipocyte hypertrophy in ob/ob mice. The Clock mutation also normalized tissue PAI-1 antigen levels in the liver but not in the adipose tissue of ob/ob mice. CONCLUSION: These observations suggest that CLOCK is involved in obesity-induced disordered fibrinolysis by regulating PAI-1 gene expression in a tissue-dependent manner. Furthermore, it appears that obesity-induced PAI-1 production in adipose tissue is not closely related to systemic PAI-1 increases in vivo.

Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression.

Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2-deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild-type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2-deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2-deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI-1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI-1 levels in Cry1/2-deficient mice remained equivalent to the peak levels of those in wild-type mice at both 9:00 and 21:00. Circadian changes in plasma PAI-1 levels seemed to be regulated at the level of gene expression, because the plasma PAI-1 levels in Clock mutant and Cry1/2-deficient mice were closely correlated with the level of PAI-1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild-type mice, and continuously higher in Clock mutant than in wild-type or Cry1/2-deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t-PA), plasma levels and mRNA levels of plasminogen, and plasma levels of alpha2 plasmin inhibitor (alpha2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.

Aberrant regulation of imprinted gene expression in Gtl2lacZ mice.

The imprinted region on mouse distal chromosome 12 covers about 1 Mb and contains at least three paternally expressed genes (Pegs: Peg9/Dlk1, Peg11/Rtl1, and Dio3) and four maternally expressed genes (Megs: Meg3/Gtl2, antiPeg11/antiRlt1, Meg8/Rian, and Meg9/Mirg). Gtl2(lacZ) (Gene trap locus 2) mice have a transgene (TG) insertion 2.3 kb upstream from the Meg3/Gtl2 promoter and show about 40% growth retardation when the TG-inserted allele is paternally derived. Quantitative RT-PCR experiments showed that the expression levels of Pegs in this region were reduced below 50%. These results are consistent with the observed phenotype in Gtl2lacZ mice, because at least two Pegs(Peg9/Dlk1 and Dio3) have growth-promoting effects. The aberrant induction of Megs from silent paternal alleles was also observed in association with changes in the DNA methylation level of a differentially methylated region (DMR) located around Meg3/Gtl2 exon 1. Interestingly, a 60 approximately 80% reduction in all Megs was observed when the TG was maternally derived, although the pups showed no apparent growth or morphological abnormalities. Therefore, the paternal or maternal inheritance of the TG results in the down-regulation in cis of either Pegs or Megs, respectively, suggesting that the TG insertion influences the mechanism regulating the entire imprinted region.

Endoscope disinfection using chlorine dioxide in an automated washer-disinfector.

Although 2% glutaraldehyde is often the first-line agent for endoscopic disinfection, its adverse reactions are common among staff and it is less effective against certain mycobacteria and spore-bearing bacteria. Chlorine dioxide is a possible alternative and an automated washer-disinfector fitted with this agent is currently available. This study was conducted to evaluate the effectiveness of chlorine dioxide in endoscopic disinfection after upper gastrointestinal examination. In vitro microbicidal properties of chlorine dioxide solutions were examined at high (600 ppm) and low (30 ppm) concentrations against various microbes including Pseudomonas aeruginosa, Helicobacter pylori, Mycobacterium avium-intracellulare and Bacillus subtilis in the presence or absence of bovine serum albumin (BSA). Immediately following endoscopic procedures and after application to the automated reprocessor incorporating chlorine dioxide at 30 ppm for 5 min, endoscopic contamination with infectious agents, blood, H. pylori ureA gene DNA and HCV-RNA was assessed by cultivation, sensitive test tape, polymerase chain reaction (PCR) and reverse transcriptase-PCR analysis, respectively. Chlorine dioxide at 30 ppm has equivalent microbicidal activity against most microbes and faster antimicrobial effects on M. avium-intracellulare and B. subtilis compared with 2% glutaraldehyde, but contamination with BSA affected the microbicidal properties of chlorine dioxide. Endoscopic contamination with microbes, blood and bacterial DNA was eliminated after application of the automated reprocessor/chlorine dioxide system. Thus, chlorine dioxide is a potential alternative to glutaraldehyde. The use of automated reprocessors with compatibility to chlorine dioxide, coupled with thorough pre-cleaning, can offer effective, faster and less problematic endoscopic disinfection.

Detection of antibodies to HTLV-I and -III in sera from Japanese hemophiliacs.

Sera from 50 Japanese hemophiliacs were screened for antibodies to human T-lymphotropic retrovirus types I and III (HTLV-I and -III). As a whole, antibody to HTLV-I, antibody to HTLV-III, and antibodies to HTLV-I and -III were detected in sera from 2, 17, and 6 hemophiliacs, respectively. Among them, two hemophiliacs developed acquired immunodeficiency syndrome who were positive for both antibodies to HTLV-I and -III in sera. All of the others were asymptomatic. Most of the blood products transfused into these hemophiliacs were imported from abroad, whence the source of HTLV-III infection presumably originated. However, since quite a high percentage of these antibody-positive hemophiliacs was positive for antibody to HTLV-I, even though they are native residents in HTLV-I nonendemic areas of Japan, some special factors may have participated in HTLV-I infection. These special factors should be investigated in the future.

Effect of growth hormone on the translocation of GLUT4 and its relation to insulin-like and anti-insulin action.

To elucidate the effect of growth hormone (GH) on the insulin signal transduction pathway leading to the translocation of glucose transporter-4 (GLUT4), we constructed Chinese hamster ovary cells that overexpressed GH receptor and GLUT4. Treatment with GH triggered GLUT4 translocation, and this translocation was completely inhibited by wortmannin. GH-induced GLUT4 translocation reached a maximum level after 30 min, and then gradually decreased and returned to the basal level after 2 h. Tyrosine phosphorylation of JAK2 also became maximal after 30 min and then gradually decreased. In contrast, GLUT4 translocation remained unchanged for 2 h after insulin treatment, and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) also remained constant for up to 2 h. Chronic GH treatment had almost no effect on insulin-stimulated Akt kinase activation and GLUT4 translocation. These results suggest that GH and insulin translocate GLUT4 in a similar manner, at least in part, and the difference in translocation depends on the difference in the tyrosine phosphorylation of JAK2 and IRS-1. The anti-insulin action of GH after chronic GH treatment does not appear to be mainly due to the inhibition of GLUT4 translocation.

Increased adipose expression of the uncoupling protein-3 gene by thiazolidinediones in Wistar fatty rats and in cultured adipocytes.

Uncoupling protein (UCP) 3 and UCP2, mitochondrial carrier proteins dissipating electrochemical gradient across the mitochondrial inner membrane, have been implicated in the regulation of energy metabolism. The UCP3 gene is expressed abundantly in the skeletal muscle, while the UCP2 gene is detected in the white adipose tissue (WAT) with diffuse localization throughout the body. Uncoupling of electron transport and ATP synthesis has been reported to increase glucose uptake, suggesting that UCP may be involved in glucose metabolism. Thiazolidinediones (TZDs), which are insulin-sensitizing agents for NIDDM, have been reported to increase energy expenditure. To elucidate the pathophysiologic significance of UCP3 and UCP2 in the effect of TZDs on glucose metabolism and energy expenditure, we examined their basal mRNA levels in the WAT, brown adipose tissue (BAT), and skeletal muscle from Wistar fatty rats, a rat model of NIDDM and obesity with leptin receptor defect, and investigated expression of the genes encoding UCP3 and UCP2 in Wistar fatty rats and in Wistar lean rats with 2-week oral administration of 3 mg x kg(-1) x day(-1) pioglitazone, a TZD derivative. Basal UCP3 mRNA levels were significantly lower (38 +/- 8, 45 +/- 13, and 76 +/- 6%) in the retroperitoneal WAT, BAT, and skeletal muscle from Wistar fatty rats than in those from Wistar lean rats, while basal UCP2 mRNA levels were significantly higher by 2.1-, 1.8-, and 2.5-fold in the subcutaneous WAT, retroperitoneal WAT, and BAT from Wistar fatty rats, respectively, than in those from Wistar lean rats. In pioglitazone-treated Wistar fatty rats, UCP3 mRNA levels were significantly increased by 2.1-, 2.0-, and 1.6-fold in the epididymal WAT, retroperitoneal WAT, and BAT, respectively, as compared with those in nontreated fatty rats. In pioglitazone-treated lean rats, UCP3 mRNA levels were significantly increased by 1.3-fold in the BAT as compared with those in nontreated lean rats. No significant change of UCP2 mRNA levels was observed in pioglitazone-treated fatty and lean rats. In addition, to examine the direct effect of TZDs on adipocytes, we examined the regulation of UCP3 and UCP2 gene expression using the primary culture of rat mature adipocytes from Sprague-Dawley rats. In rat cultured mature adipocytes, UCP3 mRNA levels were increased in a dose-responsive manner by 10(-5) to 10(-4) mol/l pioglitazone, while there was no significant change of UCP2 mRNA levels. These results clearly demonstrate that UCP3 gene expression is upregulated by TZDs in the WAT and BAT in Wistar fatty rats, an obese model with leptin receptor defect, and that adipose UCP3 gene expression is increased in response to TZDs in vitro. The present study suggests the involvement of UCP3 in the effects of TZDs on energy and glucose metabolism.

Comparison of GAD and ICA512/IA-2 antibodies at and after the onset of IDDM.

OBJECTIVE: Longitudinal changes in GAD antibody (Ab) and ICA512/IA-2 (ICA512) Ab were examined in relation to age at the onset of diabetes and autoimmunity against the thyroid gland. RESEARCH DESIGN AND METHODS: GADAb, ICA512Ab, and antithyroid autoantibody were examined at onset in 40 juvenile-onset IDDM patients (17 males, 23 females, age at onset 9.4 +/- 4.1 years, range 1.7-20). To assess the changes in antibody levels, 29 patients were followed up with sequential serum samples for up to 5 years. RESULTS: At onset, GADAb, ICA512Ab, and antithyroid autoantibody (thyroglobulinAb or thyroid peroxidaseAb) were found in 70, 58, and 25% of the 40 patients, respectively. Prepubertal patients (n = 21) had a significantly higher prevalence and index of ICA512Ab compared with pubertal patients (n = 19) (76 vs. 37%, P = 0.012, and 2.43 +/- 2.36 vs. 0.66 +/- 1.23, P = 0.011), while GADAb was more prevalent in pubertal patients (57 vs. 84%, P = 0.09). A longitudinal analysis of GADAb and ICA512Ab showed that GADAb levels declined more slowly than those of ICA512Ab (P = 0.008). Patients with continuous extremely high levels of GADAb also had high levels of antithyroid autoantibody. CONCLUSIONS: The measurement of ICA512Ab is useful in prepubertal patients, who often show rapid progression of the disease. The presence of autoimmunity against thyroid gland seems to influence the GADAb level but not the ICA512Ab level.

Efficacy of recombinant human granulocyte colony-stimulating factor on neutropenia in patients with AIDS.

The efficacy of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on neutropenia was evaluated in 14 patients with AIDS and AIDS-related complex (ARC). In all patients, including 11 neutropenic patients, 100 or 200 micrograms/m2 of rhG-CSF significantly increased the neutrophil counts. The response was greater in patients with higher neutrophil counts before the treatment, and was also dose-dependent. Although the effect seemed to be less potent, the agent also increased the neutrophil counts even when zidovudine (azidothymidine, AZT) and other myelosuppressive antiviral agents were administered simultaneously. These observations indicate that rhG-CSF may be beneficial in preventing and treating some secondary infections, and will make it easier to continue therapy with antiviral agents in patients with AIDS or ARC.

Infiltration of hematogenous lineage cells into the demyelinating central nervous system of twitcher mice.

Infiltration of hematogenous lineage cells into the central nervous system (CNS) was investigated in the twitcher mouse, a murine model of globoid cell leukodystrophy in human. The hematogenous cells were selectively labeled following intraperitoneal injection of rhodamine isothiocyanate (RhIc). The frequency of detecting Rhlc-labeled cells (Rhlc+ cells) in the twitcher CNS varied with age. RhIc+ cells were hardly detected when injection was made prior to the postnatal day (PND) 30. The number of Rhlc' cells increased thereafter peaked at PND 35-38 and declined drastically at PND 40-45. The majority of RhIc+ cells were distributed in white matter of the CNS that correlated well with the areas of demyelination and of increased microglia/macrophage population described in our earlier studies. Almost all Rhlc+ cells were double-labeled with antibody for Mac-1 and also with MHC class II. Some small cells double-labeled with RhIc and antibodies for CD4, CD8, or IL-2R were also identified. By RT-PCR, the expression of monocyte chemoattractant protein- (MCP-1) mRNA increased drastically at PND 30, peaked at PND 35, and decreased gradually after PND 40. This pattern of mRNA changes correlated well with the dynamic pattern of the infiltration of hematogenous cells into the CNS, suggesting a role of chemokine(s) in the cellular infiltration in the twitcher brain. The expression of IL-10 mRNA also increased gradually. IL-10 is a cytokine inhibitory factor and a major regulator in suppressing the inflammatory response. Thus, our results indicated that hematogenous lineage cells infiltrated in the CNS of twitcher mice, and that MCP-1 and IL-10 may play an important role in regulating the cellular recruitment.

Expression of immune-related molecules is downregulated in twitcher mice following bone marrow transplantation.

Twitcher (twi/twi) is a murine model of a human genetic demyelinating disease, globoid cell leukodystrophy (Krabbe disease). The affected mice usually die before reaching age 45 days, having demyelination associated with extensive glial activation. The twi/twi mice that receive wild-type bone marrow transplantation (BMT) survive up to 3 times longer with improved pathology. We hypothesize that immune-related molecules such as cytokines and chemokines are partly responsible for the demyelination in twi/twi, and that the decrease in the expression of such molecules following BMT contributes to clinico-pathological improvement. Cells expressing TNF-alpha, MCP-1, and MIP-1beta were conspicuous in the twi/twi CNS accompanied by infiltration of Ia+ and CD8+/CD3- hematogenous cells. These cells decreased gradually after BMT TNF-alpha mRNA and mRNA of C-C chemokine families, including MCP-1, IP-10, MIP-1alpha, MIP-1beta, and RANTES, were upregulated in the twi/twi CNS but downregulated gradually following BMT. In twi/twi that survived to 20 wk of age, cells expressing TNF-alpha, MCP-1, MIP-1beta, Ia, or CD8 were hardly detected and pathology was clearly improved. These results are consistent with the hypothesis that cytokine expression in glial cells contributes (to some extent) to the pathogenesis of demyelinating lesions in the twi/twi mice.

Development changes in long-form leptin receptor expression and localization in rat brain.

The expression and localization of long-form leptin receptor (OB-Rb) were studied immunocytochemically in the brain of fetal and adult rats using a polyclonal antibody that specifically recognized OB-Rb. At 14 days of gestation, immunoreactive cells were observed in the ventricular layer, which contains premature neuronal cells. At 18 days of gestation, they were weakly stained but obvious in the paraventricular nucleus (PVN), and ependymal cells also showed immunoreactivity. At birth, the immunoreactivity of OB-Rb in the PVN seemed to be much lower than that in adult rats and remained low during the suckling period. Considering the presence of neuroendocrine and structural neuronal abnormalities in Lepob/Lepob mice with genetic leptin deficiency, these results suggest that the expression of OB-Rb in premature neuronal cells may have some function, and that the regulation of energy balance by leptin through hypothalamic regions, such as PVN, may not yet be developed in the perinatal period.

Evidence that the FSH receptor itself is not a calcium channel.

FSH has been shown to stimulate the uptake of calcium in cultured rat Sertoli cells, resulting in an increase in cytosolic calcium concentrations. One possibility which has been put forth is that the FSH receptor per se may act as a calcium channel. This is all the more tantalizing given the proposed structure of this receptor which, like all other G protein-coupled receptors, is thought to have the putative transmembrane helices forming a bundle-like structure in the plasma membrane. To test whether the FSH receptor could function as a calcium channel, we performed whole-cell voltage clamp experiments on 293 and 293F(wt1) cells, which are a clonal line of 293 cells expressing high levels of rat FSH receptors. The 293 cells, which do not express FSH receptors, were found to lack any detectable inward calcium currents, and therefore, serve as an excellent model for transfecting with potential calcium conducting FSH receptors. When the 293F(wt1) cells were then tested, no inward calcium currents could be detected in either control or FSH-stimulated cells. These results suggest that the FSH receptor itself is not a calcium channel and, therefore, FSH must be stimulating endogenous calcium channels in rat Sertoli cell plasma membranes.