Differential regulation of the apoptotic machinery during megakaryocyte differentiation and platelet production by inhibitor of apoptosis protein Livin.

Livin is a member of the inhibitor of apoptosis proteins (IAP) family of intracellular antiapoptotic proteins that act by binding and inhibiting caspases. Upon strong apoptotic stimuli, it is then specifically cleaved by caspases to produce a truncated protein (tLivin) with a paradoxical proapoptotic activity. Intriguingly, we have detected robust protein levels of Livin in normal mature bone marrow megakaryocyte (MK) and platelets. To evaluate the potential role of Livin in thrombopoiesis, we used the human BCR-ABL+ cell line, LAMA-84, and cord blood CD34+ cells to induce differentiation toward MKs. Upon differentiation, induced by phorbol myristate acetate and concurrent with increase in Livin protein expression, LAMA-84 cells formed functional platelet-like particles. Livin overexpression in CD34+ progenitor cells induced higher endoreplication in the MKs generated. Furthermore, overexpression of Livin increased the ability of both primary MKs and differentiated LAMA-84 cells to produce functional platelets. In the differentiated LAMA-84 cells, we observed accumulation of proapoptotic tLivin concomitant with increased caspase-3 activity. Downregulation of Livin with small interfering RNA in both leukemic and primary MK cells decreased their ability to produce functional platelets. We suggest that Livin has a role in thrombopoiesis by regulating the apoptotic and antiapoptotic balance in MK endoreplication and platelet production.

Insulin in human milk: postpartum changes and effect of gestational age.

OBJECTIVE: To determine if human milk insulin (HMI) concentrations are affected by gestational age and postnatal age. DESIGN AND SETTING: An observational study carried out in a level III neonatal intensive care unit. Insulin concentrations were determined in human milk of 90 parturient mothers who delivered between 30 and 41 weeks gestation. Samples were collected on days 3 and 10 after delivery. RESULTS: HMI concentrations for mothers of preterm infants were not significantly different from those of full term infants, on either day 3 or 10 post partum. When results for all 90 mothers were pooled, regardless of gestational age, HMI concentration fell significantly from day 3 to day 10 (50.1 (34.6) v 41.1 (28.5) microU/ml; p = 0.01; mean (SD)). However, this decrease was only significant for mothers delivering at term (37-41 weeks). CONCLUSIONS: HMI concentrations were not influenced by gestational age at delivery. They decreased post partum, mainly in mothers of term infants. The postnatal changes in HMI concentrations and the effects of oral insulin on the immature intestinal mucosa warrant further investigation.

TGF-beta-induced apoptosis is mediated by the adapter protein Daxx that facilitates JNK activation.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.

Importance of insulin content in infant diet: suggestion for a new infant formula.

Oral insulin promotes intestinal maturation and may prevent diabetes in animal models. The aim of this study was to evaluate the concentration of insulin in human milk and in different infant formulas. Our results show that the concentration of insulin in human milk is significantly higher (60.23 +/- 41.05 microU/ml mean +/- SD) compared with cow's milk (16.32 +/- 5.98 microU/ml mean +/- SD) and that insulin is hardly detectable in infant formulas. We propose the addition of human insulin to infant formula to match its composition more closely to human milk.

Intraoperative radioisotope sentinel lymph node mapping in non-small cell lung cancer.

BACKGROUND: Lymph node metastases are the most significant prognostic factor in localized non-small cell lung cancer (NSCLC). Nodal micrometastases may not be detected. Identification of the first nodal drainage site (sentinel node) may improve detection of metastatic nodes. We performed intraoperative Technetium 99m sentinel lymph node (SN) mapping in patients with resectable NSCLC. METHODS: Fifty-two patients (31 men, 21 women) with resectable suspected NSCLC were enrolled. At thoracotomy, the primary tumor was injected with 2 mCi Tc-99. After dissection, scintographic readings of both the primary tumor and lymph nodes were obtained with a handheld gamma counter. Resection with mediastinal node dissection was performed and findings were correlated with histologic examination. RESULTS: Seven of the 52 patients did not have NSCLC (5 benign lesions, and 2 metastatic tumors) and were excluded. Forty-five patients had NSCLC completely resected. Mean time from injection of the radionucleide to identification of sentinel nodes was 63 minutes (range 23 to 170). Thirty-seven patients (82%) had a SN identified; 12 (32%) had metastatic disease. 35 of the 37 SNs (94%) were classified as true positive with no metastases found in other intrathoracic lymph nodes without concurrent SN involvement. Two inaccurately identified SNs were encountered (5%). SNs were mediastinal (N2) in 8 patients (22%). CONCLUSIONS: Intraoperative SN mapping with Tc-99 is an accurate way to identify the first site of potential nodal metastases of NSCLC. This method may improve the precision of pathologic staging and limit the need for mediastinal node dissection in selected patients.

Prolactin-induced cell proliferation in PC12 cells depends on JNK but not ERK activation.

The effects of pituitary and extrapituitary prolactin include cellular proliferation and differentiation. PC12 cells was used as a model to delineate respective signaling of prolactin. Prolactin acted as a mitogen for undifferentiated PC12 cells, as measured by significant increases in bromodeoxyuridine incorporation and in cell numbers, with an efficacy equal to epidermal growth factor. Both the long and short form of the prolactin receptor was expressed, yet only the long isoform was tyrosine-phosphorylated upon agonist binding. Functional prolactin receptor signaling was further demonstrated in the activation of JAK2 and phosphorylation activation of the transcription factors Stat1, -3, and -5a. Surprisingly, prolactin stimulated a sustained activation of Raf-B, without activation of the MAP kinases ERK1 or -2. Instead, in solid phase kinase assays using a glutathione S-transferase-c-Jun fusion protein (amino acids 1-79) as the substrate, a significant activation of the mitogen-activated protein Janus kinase (c-Jun N-terminal kinase; JNK) was observed. The prolactin-induced activation of JNK was prolonged and accompanied by a significant increase in c-Jun mRNA abundance and c-Jun protein synthesis. Moreover, analysis of bromodeoxyuridine incorporation at the single cell level revealed that epidermal growth factor-dependent incorporation was inhibited by PD98059 and independent of SB203580, whereas prolactin-induced incorporation was ERK and mitogen-activated protein kinase p38 independent but was abolished with JNK inhibition by 30 microm SB203580. Our studies suggest that prolactin may have a role in the growth of PC12 cells, where it stimulates concurrent mitogenic and differentiation-promoting signaling pathways.

Sentinel lymphadenectomy for breast cancer: experience with 180 consecutive patients: efficacy of filtered technetium 99m sulphur colloid with overnight migration time.

BACKGROUND: Axillary node status remains the most important prognostic indicator of survival in breast cancer patients. Only 25% to 35% of patients having standard level I/II axillary dissection have involved nodes, yet all accept the potential for morbidity after the operation. This study was conducted to assess whether status of the sentinel node(s) was an accurate predictor of the presence of metastatic disease in axillary or internal mammary nodes. STUDY DESIGN: In 180 patients, technetium 99m sulphur colloid was injected in a 4-quadrant peritumoral distribution. During the first phase of the study, 72 patients had sentinel node excision followed by a level I/II axillary dissection. During the second phase of the study, 108 patients had sentinel node excision and only those with positive nodes had completion axillary dissection. Nodes were examined after formalin fixation by taking 10 sections at 20-microm intervals and staining with hematoxylin-eosin. RESULTS: Sentinel nodes were found in 162 (90%) of 180 patients. The mean number of sentinel nodes examined was 3.1. Of the 162 patients with successful lymphatic mapping, positive sentinel nodes were found in 44 (27%). In 23 (66%) of 35 patients with positive sentinel nodes who had a completion level I/II axillary dissection, the sentinel nodes were the only positive nodes. The concurrent negative predictive value was 4% in the first 72 patients who had completion axillary dissection after sentinel node excision, and 2% for the entire series. With evolution of technique, identification of sentinel nodes with radiolabeled colloid was successful in 97% of the last 100 patients. CONCLUSIONS: Because the concurrent negative predictive value was low, sentinel node excision appeared to accurately identify node status, potentially avoiding the need for standard level I/II axillary dissection in sentinel node-negative patients.

Insulin-degrading enzyme does not require peroxisomal localization for insulin degradation.

Although considerable evidence implicates insulin-degrading enzyme (IDE) in the cellular metabolism of insulin in many cell types, its mechanism and site of action are not clear. In this study, we have examined the relationship between insulin-degrading enzyme's peroxisomal location and its ability to degrade insulin by mutation of its peroxisomal targeting signal (PTS), the carboxy terminal A/S-K-L tripeptide. Site-directed mutagenesis was used to destroy the peroxisomal targeting signal of human insulin-degrading enzyme by changing alanine to leucine (AL.pts), leucine to valine (LV.pts), or by deleting the entire tripeptide (DEL.pts). The alanine or leucine mutants, when expressed in COS cells, were indistinguishable from wild-type insulin-degrading enzyme with respect to size (110 kDa), amount of immunoreactive material, ability to bind insulin, in vitro activity, and cellular degradation of insulin. In contrast, the deletion mutant was shorter in size (approximately 0 kDa) and unable to bind the hormone. Thus, although the tripeptide at insulin-degrading enzyme's carboxy terminus appeared to confer enzyme stability, the conserved sequence was not required for insulin degradation. Finally, an immunocytofluorescence study showed that, whereas a significant amount of the wild-type protein was localized in peroxisomes, none of the peroxisomal targeting mutants could be detected in these organelles. These findings indicate that insulin-degrading enzyme does not require peroxisomal localization for insulin degradation and suggest that this enzyme has multiple cellular functions.

Stress hyperglycemia and the risk for the development of type 1 diabetes.

Transient hyperglycemia during acute illness may represent the earliest clinical sign of impaired beta cell function. This study sought to characterize the clinical presentation of patients with stress hyperglycemia and to determine the prevalence of immunologic and endocrinologic markers associated with prediabetes. Thirty-six children were studied. They were referred to us for routine evaluation after an episode of hyperglycemia during severe intercurrent illness. Immunologic markers (insulin autoantibodies and islet cell autoantibodies) and intravenous glucose tolerance test for evaluation of first phase insulin secretion rate were performed in all participants. Islet cell autoantibodies were negative in all patients. In eight patients, the first phase insulin response was below the first percentile (46 microU/ml) at the first determination. Insulin autoantibodies were positive in another three children (> 60 nU/ml). Twelve to sixteen months later, all children were re-evaluated and all had normal results. None of the patients developed diabetes during the study (mean 3.2 years). Our data support the idea that episodes of hyperglycemia during severe illness without additional risk factors are a minimal risk factor, if any, for future development of IDDM.

Differential regulation of phenylethanolamine N-methyltransferase expression in two distinct subpopulations of bovine chromaffin cells.

Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.

A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma.

In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Differences in the composition of chromaffin granules in adrenaline and noradrenaline containing cells of bovine adrenal medulla.

Several constituents of chromaffin granules were quantitatively determined in noradrenaline and adrenaline cells purified from bovine adrenal medulla. As far as secretory peptides are concerned noradrenaline granules contained slightly more secretogranin II, but much less chromogranin A than adrenaline granules. This can be explained by the dependence of the biosynthesis of chromogranin A on corticosteroids. Proteolytic processing of chromogranin A and secretogranin II was higher in noradrenaline cells which was paralleled by a higher content of the prohormone convertase PC2. Noradrenaline granules also contained a higher concentration of the vesicular monoamine transporter (vMAT2). No differences were found for dopamine beta-hydroxylase, prohormone convertase PC1, carboxypeptidase H and synaptophysin. These results indicate that the secretory cocktail of peptides released from these cells differs significantly between adrenaline and noradrenaline storing cells.

Inhibition of lymphocyte activation by catecholamines: evidence for a non-classical mechanism of catecholamine action.

The effects of noradrenaline and other adrenergic agonists on lymphocyte activation were studied. Spleen and thymus cells from BALB/c mice were stimulated by mitogens and lymphocyte activation was monitored by measuring the incorporation of [methyl-3H]thymidine into DNA. Noradrenaline, adrenaline, isoproterenol and dopamine all inhibited the activation of spleen and thymus cells by concanavalin A, a T-cell specific mitogen, and the activation of spleen cells by lipopolysaccharide, a T-independent B-cell mitogen. The various catecholamines were approximately equipotent, having IC50 of approximately 10 microM. alpha-adrenergic agonists (phenylephrine, clonidine) did not inhibit lymphocyte activation. Noradrenaline, adrenaline and isoproterenol also inhibited DNA synthesis in S49 T lymphoma cells. The effects of adrenergic receptor antagonists on lymphocyte function were also studied. The inhibition of lymphocyte activation by catecholamines could not be reversed by antagonists to beta-adrenergic receptors (propranolol), alpha-adrenergic receptors (phentolamine), or dopaminergic receptors (haloperidol). Experiments with human peripheral blood leucocytes revealed that, as with murine cells, the beta-adrenergic antagonists propranolol and nadalol did not affect the catecholamine-mediated inhibition of lymphocyte activation. Although lymphocytes contain beta-adrenergic receptors that are coupled to adenylyl cyclase activity, catecholamines appear to inhibit murine lymphocyte activation by a mechanism that is independent of these or other classical adrenergic receptors.

Neurotransmitter suppression of the in vitro generation of a cytotoxic T lymphocyte response against the syngeneic MOPC-315 plasmacytoma.

We have previously shown that, as a consequence of low-dose melphalan (L-phenylalanine mustard (L-PAM) therapy, the hitherto immunosuppressed spleen cells from BALB/c mice bearing a large MOPC-315 tumor (in contrast to spleen cells from normal mice) acquire the ability to generate a greatly enhanced anti-MOPC-315 cytotoxic T lymphocyte (CTL) response upon in vitro stimulation with MOPC-315 tumor cells. Here we show that the catecholamines norepinephrine, epinephrine, and isoproterenol suppressed the in vitro generation of anti-MOPC-315 cytotoxicity by spleen cells from mice that had just completed the eradication of a large MOPC-315 tumor following low-dose L-PAM therapy (L-PAM TuB spleen cells), as well as by spleen cells from normal mice. In contrast to the marked suppression obtained with catecholamines, the cholinergic agonist carbachol had no effect on the in vitro generation of splenic anti-MOPC-315 cytotoxicity. The inhibitory effect of the catecholamines was "mimicked" by the membrane penetrating analog of cAMP, dibutyryl-cAMP, and by cholera toxin at concentrations that stimulate the endogenous production of cAMP. The beta-adrenergic receptor antagonist propranolol did not block norepinephrine-induced inhibition of the generation of anti-MOPC-315 cytotoxicity by either normal or L-PAM TuB spleen cells. Since the curative effectiveness of low-dose L-PAM therapy for MOPC-315 tumor bearers requires the participation of CD8+ T cells that exploit a CTL response in tumor eradication, it is conceivable that norepinephrine may reduce the therapeutic outcome of low-dose chemotherapy by inhibiting the acquisition of CTL activity.

Glucocorticoids enhance histamine-evoked catecholamine secretion from bovine chromaffin cells.

The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC50 for dexamethasone was approximately 1 nM. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H1 histamine receptor antagonist mepyramine but was not affected by the H2 antagonist cimetidine; thus, dexamethasone appeared to enhance an H1 receptor-mediated process. In the absence of glucocorticoids, H1 receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H1 receptor mRNA levels in both cell types. The increased expression of H1 receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.

Identification of zinc ligands of the insulin-degrading enzyme.

The insulin degrading enzyme (IDE), a nonlysosomal enzyme involved in the metabolism of internalized insulin, is a member of a new family of metalloproteases which has an HXXEH active site motif. We have previously shown that both His108 and Glu111 within the HXCEH domain of human IDE are necessary for catalytic activity. Comparison to the prototypic zinc metalloprotease thermolysin, which contains an inversion of this motif, would predict that His112, as well as a downstream glutamate, serves as the second and third zinc ligands of IDE. To examine the role of His112, we mutated this residue to glutamine, leucine, or arginine. To identify a downstream zinc ligand, we substituted a glutamine for glutamate at either Glu182 or Glu189, both of which are conserved in human, rat, and Drosophila IDE. Vectors containing wild type or mutant IDE genes were transfected into COS cells, and the enzymes were analyzed for insulin degradation, insulin cross-linking, and zinc binding. Our results suggest that His108, His112, and Glu189 are the zinc ligands of human IDE, and Glu182 can influence zinc binding. In addition to a catalytic role, zinc binding to these residues appears to play a role in stabilizing the structure of the enzyme.

1,25(OH)2 vitamin D3 activates PKC-alpha in Caco-2 cells: a mechanism to limit secosteroid-induced rise in [Ca2+]i.

Our laboratory recently reported that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly increases the breakdown of membrane phosphoinositides, raises intracellular calcium concentration ([Ca2+]i), and translocates protein kinase C (PKC) from the cytosolic to the particulate fraction of Caco-2 cells. In the present experiments, we found that Caco-2 cells contained predominantly the alpha- and zeta-isoforms of PKC, with minimally detectable amounts of PKC-beta and -epsilon by Western blotting. 1,25(OH)2D3 and the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) each caused time-dependent translocations of PKC-alpha, but not PKC-zeta. TPA treatment of these cells for 24 h induced a significant concentration-dependent downregulation of PKC-alpha, but not PKC-zeta. Since PKC inhibits phospholipase C-induced mobilization of Ca2+ in other cells, we examined the effects of staurosporine and H-7, PKC inhibitors, and TPA on 1,25(OH)2D3-stimulated increase in [Ca2+]i. As previously demonstrated by our laboratory, 1,25(OH)2D3 caused a biphasic increase in [Ca2+]i, with an initial elevation (transient phase) followed by a sustained increase (plateau phase). We previously demonstrated that the transient phase is mediated, at least in part, by an increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] stimulated by the secosteroid. Acute pretreatment with staurosporine or H-7 caused a significant stimulation, whereas acute TPA pretreatment caused a significant inhibition of the 1,25(OH)2D3-induced increase in the transient phase of [Ca2+]i. Preincubation of Caco-2 cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxy-methyl ester (BAPTA-AM) abolished both the rise in [Ca2+]i and the increase in particulate-associated PKC-alpha stimulated by 1,25(OH)2D3. Moreover, downregulation of PKC-alpha by chronic TPA treatment significantly augmented the transient phase of the 1,25(OH)2D3-stimulated rise in [Ca2+]i but had no effect on the 1,25(OH)2D3-induced change in Ins(1,4,5)P3 concentration. Furthermore, in these PKC-alpha downregulated cells staurosporine no longer increased the secosteroid-stimulated transient rise in [Ca2+]i. These results indicate that 1,25(OH)2D3, which increases [Ca2+]i and diacylglycerol, activates PKC-alpha, but not PKC-zeta. The alpha-isoform, in turn, limits the secosteroid-stimulated rise in [Ca2+]i, at a step distal to Ins(1,4,5)P3 accumulation in Caco-2 cells.

Tetraethylammonium selectively stimulates secretion from noradrenergic bovine chromaffin cells.

1. The effects of tetraethylammonium chloride (TEA) on catecholamine secretion from primary cultures of noradrenaline-rich (noradrenergic) and adrenaline-rich (adrenergic) bovine chromaffin cells were studied. TEA stimulated catecholamine secretion from both cell types but was a much more effective secretory stimulus for noradrenergic cells. 2. TEA-induced catecholamine secretion was dependent on extracellular Ca2+, was partially inhibited by nifedipine and by tetrodotoxin, and was potentiated by ouabain. Other K+ channel blocking agents including 4-aminopyridine, glibenclamide, and tolbutamide did not stimulate catecholamine secretion. 3. TEA had no effect on Ca(2+)-induced secretion from digitonin-permeabilized chromaffin cells. 4. TEA presumably evokes secretion by inhibiting K+ channels, depolarizing chromaffin cells, and activating voltage-gated Ca2+ channels in the cells. Noradrenergic cells appear to be more sensitive to K+ channel inhibition than are adrenergic cells. 5. The secretory response of the chromaffin cells to TEA increased with time in culture. 6. In addition to being a more effective secretagogue in noradrenergic cells, TEA was also more effective in stimulating catecholamine synthesis in these cells.