Intracellular localization of calcium in the chromaffin cells of the rat adrenal medulla

The ulstrastructural localization of calcium in the chromaffin cells of the adrenal medulla was carried out by using potassium pyroantimonate during fixation. Calcium-containing deposits were either diffuse within the cytoplasm or associated with membrane-bounded organelles. Variable amounts of precipitates were found within the nucleus and in the Golgi complex. However, the major sites of calcium antimonate deposits were the secretory granules and the mitochondria. The morphological identification of calcium-storing organelles in the adreno-medullary cells may be useful in evaluating the involvement of such intracellular compartments during the secretory process.     

Neural stem cells: from fly to vertebrates

Our goal in this review is to explore the relationship between Drosophila and vertebrate neural stem cell development by comparing progress in each system with the aim of answering several central questions in stem cell biology: (a) How are stem cells formed? (b) Do stem cells divide symmetrically or asymmetrically? (c) How is stem cell fate maintained? (d) How is stem cell differentiation initiated? (e) How are different stem cell fates determined? (f) How "plastic" are different neural stem cell fates? (g) How do neural stem cells produce different progeny? and (h) What regulates stem cell proliferation versus quiescence? Not surprisingly, research in Drosophila and vertebrate systems each have their own biases, strengths, and weaknesses; we hope that by directly comparing progress in each field, new experiments and interpretations in both vertebrate and Drosophila research will become apparent. It has become increasingly clear that vertebrates and Drosophila share many fundamental mechanisms of neurogenesis, validating a comparative approach.     

Ecdysteroidostatin from the house fly, Musca domestica

Ovaries from house flies maintained on sucrose secrete large amounts of ecdysteroid when they are cultured with ovarian ecdysteroidogenic hormone, OEH. However, ovarian ecdysteroid secretion is reduced by incubation with both OEH and the ovarian ecdysteroidostatin (OES). A partially purified OES fraction from a semi-preparative reverse phase HPLC C18 column caused a 98% inhibition of ovarian ecdysteroid secretion in vitro at a concentration of 0.8 equivalents per microliter. Ovaries can be activated to produce ecdysteroid in vivo by feeding diet containing protein to flies maintained on sucrose. Ecdysteroid secretion was inhibited when the in vivo stimulated ovaries were cultured with OES. This suggests that OES does not interfere with the OEH activation mechanism, but blocks ovarian ecdysteroid synthesis or release. Furthermore, OES inhibition is reversible and ecdysteroid secretion resumes when OES is removed. Musca OES could explain the decrease in ecdysteroid levels found in flies after mid-vitellogenesis. Both adult male and female abdomens contain OES, but OES was not transferred to females during mating. Evidence is presented that OES is not a trypsin modulating oostatic factor.     

Innervation of serotonergic medullary raphe neurons from cells of the rostral ventrolateral medulla in rats

The rostral ventral medulla has been shown to consist of three distinct subregions: the midline or raphé region, the lateral paragigantocellular-gigantocellular region and the rostro-ventrolateral reticular nucleus. All three regions have been shown to contribute to central vaso-regulation and to project towards sympathetic preganglionic neurons of the thoracic spinal cord. Therefore it is of particular interest to describe the interconnections between the three regions and to see if local afferents reach cells which have been implicated in the regulation of descending inputs. Following injections of the anterograde tract tracer Phaseolus vulgaris leucoagglutinin into the lateral paragigantocellular nucleus or the rostroventrolateral reticular nucleus, labelled axons were traced into the medullary raphé nuclei and the contralateral rostral ventrolateral medulla. Efferents originating from both regions innervated the raphé pallidus, raphé obscurus and raphé magnus. However the distribution of terminals originating from the two regions was different in the contralateral ventrolateral medulla oblongata. The data indicate that the connection between the ipsi- and contralateral equivalents of both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus are stronger than the cross-connection between the ipsi- and contralateral parts of the two different regions. In the second part of the study, the existence of direct projections from the rostroventrolateral reticular nucleus and the lateral paragigantocellular-gigantocellular region onto serotonin-immunogold-labelled cells of the ventromedial medulla were investigated. The correlated light and electron microscopic analysis revealed direct synaptic contacts between axons originating from both the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus, and serotonin-immunoreactive cells of the raphé obscurus and raphé pallidus. The results of the present light microscopic tract-tracing study revealed a different pattern of the intramedullary projection of the lateral paragigantocellular-gigantocellular region and the rostroventrolateral reticular nucleus. These data are in support of the proposed parcellation of the two cytoarchitectonically different areas of the rostral ventrolateral medulla into two functionally distinct subdivisions. Furthermore, the direct anatomical connection revealed in the present study between cells of the rostral ventrolateral and ventromedial medulla oblongata indicates the possibility that vasoregulatory effects of some cells of the rostral ventrolateral medulla oblongata might be executed via direct projections onto serotonin-immunoreactive cells of the medullary raphé nuclei.     

Intracellular calcium, currents, and stimulus-response coupling in endothelial cells

Vascular endothelium appears to be a unique organ. It not only responds to numerous hormonal and chemical signals but also senses changes in physical parameters such as shear stress, producing mediators that modulate the responses of numerous cells, including vascular smooth muscle, platelets, and leukocytes. In many cases, the initial response of endothelial cells to these diverse signals involves elevation of cytosolic Ca2+ and activation of Ca(2+)-dependent enzymes, including nitric oxide synthase and phospholipase A2. Both the release of Ca2+ from intracellular stores, most likely the endoplasmic reticulum, and the influx of Ca2+ from the extracellular space contribute to the [Ca2+]i increase. The most important trigger for Ca2+ release is inositol 1,4,5-trisphosphate, which is generated by the action of phospholipase C, a plasmalemmal enzyme activated in many cases by the receptor-G protein cascade. Ca2+ influx appears to be related to the activity of receptor-G protein-enzyme complex and to the degree of fullness of the endoplasmic reticulum but does not involve voltage-gated Ca2+ channels. The magnitude of the Ca2+ influx depends on the electrochemical gradient, which is modulated by the membrane potential, Vm. Under basal conditions, Vm is dominated by a large inward rectifier K+ current. Some stimuli, e.g., acetylcholine, have been shown to hyperpolarize Vm, thus increasing the electrochemical gradient for Ca2+, which appears to be modulated by activation of Ca(2+)-dependent K+ and Cl- currents. However, the lack of potent and specific blockers for many of the described or postulated channels (e.g., nonselective cation channel, Ca(2+)-activated Cl- channel) makes an estimation of their effect on endothelial cell function rather difficult. Possible future directions of research and clinical implications are discussed.     

Intracellular study of synaptic events related to phase-locking responses of cat cochlear nucleus cells to low frequency tones

In this study intracellular recording techniques were used to study the synaptic events related to phase-locking of cochlear nucleus cells to low frequency stimuli. A variable degree of phase-locking was noted even with units of the same low characteristic frequency. With low frequency phase-locking units an excitatory postsynaptic potential (EPSP) occurred in response to each period of the frequency stimulus, but the probability of an action potential occurring decreased as the frequency of the stimulus was raised. Complex units were described which phase-locked at lower frequencies of stimulation but did not at higher frequencies where the temporal pattern of firing to tone burst stimulation changed as well. Results are discussed as they relate to the frequency following response recorded with gross electrodes in the lower auditory pathway and the relationship to frequency coding in the auditory pathway.     

Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous, antileukemic T-cell responses

We have previously reported that leukemic dendritic cells (DC) can be generated ex vivo from myelomonocytic precursors in chronic myelogenous leukemia. In this study we report the generation of DC from acute myelogenous leukemia (AML) cells and their potent ability to stimulate leukemia-specific cytolytic activity in autologous lymphocytes. DC were generated in vitro using granulocyte-macrophage colony-stimulating factor +interleukin-4 in combination with either tumor necrosis factor-alpha or CD40 ligand (CD40L). Cells from 19 AML patients with a variety of chromosomal abnormalities were studied for their ability to generate DC. In all but 1 case, cells with the morphology, phenotypic characteristics, and T-cell stimulatory properties of DC could be generated. These cells expressed high levels of major histocompatibility complex class I and class II antigens as well as the costimulatory molecules B7-2 and ICAM-1. In three cases these cells were determined to be of leukemic origin by fluorescence in situ hybridization for chromosomal abnormalities or Western blotting for the inv(16) fusion gene product. Autologous lymphocytes cocultured with AML-derived DC (DC-AL) were able to lyse autologous leukemia targets, whereas little cytotoxicity was noted against autologous, normal cells obtained from the patients during remission. We conclude that leukemia derived DC may be useful for immunotherapy of many AML patients.     

Co-existence of P2Y-and PPADS-insensitive P2U-purinoceptors in endothelial cells from adrenal medulla

1. We have studied the effects of purinoceptor stimulation on Ca2+ signals in bovine adrenomedullary endothelial cells. [Ca2+]i was determined with the fluorescent probe fura-2 both in population samples and in single, isolated, endothelial cells in primary culture and after subculturing. 2. In endothelial cells, maintained in culture for more than one passage, several purinoceptor agonists elicited clear [Ca2+]i transient peaks that remained in the absence of extracellular Ca2+. Adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) were equipotently active, with EC50 values of 8.5 +/- 0.9 microM and 6.9 +/- 1.5 microM, respectively, whereas 2-methylthioadenosine 5'-triphosphate (2MeSATP), adenosine 5'-(alpha, beta-methylene)triphosphate (alpha, beta-MeATP) and adenosine(5')tetraphospho(5')adenosine (Ap4A) were basically inactive. Adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) was a weak agonist. The apparent potency order was UTP = ATP > ADP beta S > 2MeSATP > alpha, beta-MeATP. 3. Cross-desensitization experiments revealed that UTP or ATP, added sequentially at concentrations of maximal effect, could completely abolish the [Ca2+]i response to the second agonist. ADP beta S exerted only a partial desensitization of the response to maximal ATP, in accordance with its lower potency in raising [Ca2+]i. 4. The effect on [Ca2+]i of 100 microM ATP in subcultured cells was reduced by only 25% with 100 microM suramin pretreatment and was negligibly affected by exposure to 10 microM pyridoxalphosphate-6-azophenyl-2', 4'-disulphonic acid (PPADS). The concentration-effect curve for ATP was not significantly affected by PPADS, but was displaced to the right by a factor of 6.5 by 100 microM suramin. 5. In primary cultures, clear [Ca2+]i responses were elicited by 2MeSATP. Suramin totally and selectively blocked 2MeSATP responses, whereas UTP-evoked [Ca2+]i transients were mainly unaffected by suramin or PPADS. Over 80% of cells tested showed responses to both 2MeSATP and UTP. The [Ca2+]i response to UTP was not desensitized in the presence of 2MeSATP. 6. ATP and UTP stimulated the release of preloaded [3H]-arachidonic acid ([3H]-AA), both in the presence and in the absence of extracellular Ca2+, by approximately 135% with respect to basal levels. Suramin and PPADS enhanced, rather than inhibited, the [3H]-AA releasing effect of ATP by 2.5 times. Suramin also potentiated the effect of the calcium ionophore A23187. 7. These results indicate that endothelial cells from adrenomedullary capillaries co-express both P2Y- and P2U-purinoceptors. P2Y-purinoceptors are lost in culture with the first passage of the cells. The P2U-purinoceptor subtype present in these cells is insensitive to PPADS and thus similar to that found in aortic endothelial cells.     

Unit responses in the medulla oblongata of rabbit to changes in local and cutaneous temperature

Normally occurring neuron death and that brought about by prior removal of the peripheral target organ was studied ultrastructurally in embryonic chick ciliary ganglion in order to better understand the mechanism of cell death in this system. Before the period of cell death, all neurons in the normal ganglion developed a well-organized rough endoplasmic reticulum (RER) which coincided with peripheral synapse formation. None of the peripherally deprived neurons underwent this change, suggesting that some interaction with the periphery, possibly synapse formation, triggered them into the secretory state. Cell death in peripherally deprived neurons was signalled by nuclear changes followed by freeing of ribosomes from polysomes and RER and presumably cessation of protein synthesis. In contrast, normal cell death was brought about by dilation of the RER with eventual cytoplasmic disruption, nuclear changes appearing only secondarily. It is suggested that failure to form or maintain peripheral synapses could result in the accumulation of transmission-related proteins with consequent cisternal dilation, and eventual cell death.     

Peptide-amidating enzymes are expressed in the stellate epithelial cells of the thymic medulla

C-terminal amidation is a post-translational processing step necessary to convey biological activity to a large number of regulatory peptides. In this study we have demonstrated that the peptidyl-glycine alpha-amidating monooxygenase enzyme complex (PAM) responsible for this activity is located in the medullary stellate epithelial cells of the thymus and in cultured epithelial cells bearing a medullary phenotype, using Northern blot, immunocytochemistry, in situ hybridization, and enzyme assays. Immunocytochemical localization revealed a granular pattern in the cytoplasm of the stellate cells, which were also positive for cytokeratins and a B-lymphocyte-associated antigen. The presence of PAM activity in medium conditioned by thymic epithelial cell lines suggests that PAM is a secreted product of these cells. Among the four epithelial cell lines examined, there was a direct correlation between PAM activity and content of oxytocin, an amidated peptide. Taken together, these data provide convincing evidence that thymic epithelial cells have the capacity to generate amidated peptides that may influence T-cell differentiation and suggest that the amidating enzymes could play an important role in the regulation of thymic physiology.     

Intracellular calcium responses of circadian pacemaker neurons measured with fura-2

The circadian pacemaker in the eye of the mollusk Bulla gouldiana is located within basal retinal neurons (BRNs) that express a circadian rhythm in cell culture. Light and other depolarizing stimuli shift the phase of the pacemaker in the eye through a process that requires extracellular calcium and is blocked by Ni2+. To test directly if an influx of Ca2+ is present throughout depolarizing treatments that produce phase shifts, dissociated BRNs in cell culture were loaded with a membrane-permeable form of the calcium-sensitive dye fura-2, and then depolarized with elevated levels of extracellular K+. Calcium levels in the BRNs remained elevated during treatments with 50 mM K+ lasting 1 h, a sufficient duration to phase shift the circadian pacemaker. Lowering extracellular free Ca2+ (approx. 1.7 x 10(-7) M) during depolarization blocked the rise in intracellular Ca2+, verifying that a Ca2+ influx is required. The sustained Ca2+ elevation during depolarization was also prevented with 50 mM Ni2+, which blocks phase shifts of the rhythm to depolarization, but not with 5 mM Ni2+, which does not block phase shifts. The initial rise in [Ca2+]i in response to 50 mM K+ was largest on average during the subjective night. The results show that a critical portion of the entrainment pathway persists in pacemaker neurons during cell culture, and that the phase-shifting stimulus may depend on a prolonged Ca2+ signal.     

Clock-spiking cells not only in the eye of the fly, but also in the antenna!

Progressive pseudorheumatoid dysplasia (spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDTPA) or progressive pseudorheumatoid arthropathy of childhood (PPAC) (MIM 208.230)) is an autosomal recessively inherited skeletal dysplasia with changes in the spine similar to spondyloepiphyseal dysplasia tarda. The disease begins mostly between the ages of 2 to 8 years with progressive joint stiffness and pain, soft tissue swelling and deformities of multiple joints including the proximal interphalangeal joints of the hands. We describe a patient where a symmetric rhizomelic shortening of the extremities and a bilateral severe pes equinovarus was present at birth.     

Potassium-evoked directionally selective responses from rabbit retinal ganglion cells

Previous studies have shown that directionally selective (DS) retinal ganglion cells cannot only discriminate the direction of a moving object but they can also discriminate the sequence of two flashes of light at neighboring locations in the visual field: that is, the cells elicit a DS response to both real and apparent motion. This study examines whether a DS response can be elicited in DS ganglion cells by simply stimulating two neighboring areas of the retina with high external K+. Extracellular recordings were made from ON-OFF DS ganglion cells in superfused rabbit retinas, and the responses of these cells to focal applications of 100 mM KCl to the vitreal surface of the retina were measured. All cells produced a burst of spikes (typically lasting 50-200 ms) when a short pulse (10-50 ms duration) of KCl was ejected from the tip of a micropipette that was placed within the cell's receptive field. When KCl was ejected successively from the tips of two micropipettes that were aligned along the preferred-null axis of a cell, sequence-dependent responses were observed. The response to the second micropipette was suppressed when mimicking motion in the cell's null direction, whereas an enhancement during apparent motion in the opposite direction frequently occurred. Sequence discrimination in these cells was eliminated by the GABA antagonist picrotoxin and by the Ca(2+)-channel blocker omega-conotoxin MVIIC, two drugs that are known to abolish directional selectivity in these ganglion cells. The spatiotemporal properties of the K(+)-evoked sequence-dependent responses are described and compared with previous findings on apparent motion responses of ON-OFF DS ganglion cells.     

Interstitial cells of the medulla and the renin-angiotensin system in disorders of water-salt homeostasis

Electron microscopic studies of the intersticial cells of the renal medullar layer and the count of lipid granules in them were conducted in two experimental states accompanied by a reduction of the circulating blood volume--72 hours of water-free diet and intravenous administration of Lazyx that possesses a potent natriuretic and diuretic effect. The concentration of renin and angiotensin was determined in the plasma of the same animals, using the radioimmunoassay technique, and the juxtaglomerular apparatus of the kidneys was examined. The study has demonstrated that the reduction of the circulating blood volume is accompanied, on the one hand, by a significant elevation of the plasma concentration of renin and angiotensin, and, on the other hand, by a reduction of the number of lipid granules in the intersticial cells that is interpreted by the authors as an index of an increased synthesis of renal prostaglandins. It has been concluded that a close functional interrelashionship exists between the two hormonal systems of the kidneys that regulate the water-electrolyte homeostasis, i.e. the renin-angiotensin system and the prostaglandins system.     

Callatostatins: neuropeptides from the blowfly Calliphora vomitoria with sequence homology to cockroach allatostatins

Five neuropeptides with C-terminal amino acid sequence homology to cockroach allatostatins have been identified in the blowfly Calliphora vomitoria. Three have the same pentapeptide C-terminal amino acid sequence as allatostatin 1 of the cockroach Diploptera punctata. A hexadecapeptide designated callatostatin 1, isolated from thoracic ganglia, brains, and heads, has the sequence Asp-Pro-Leu-Asn-Glu-Glu-Arg-Arg-Ala-Asn-Arg-Tyr-Gly-Phe-Gly-Leu-NH2. Callatostatins 2 and 3 have been isolated from heads and thoracic ganglia, respectively; they comprise the last 14 and 8 residues of callatostatin 1. Callatostatin 4, isolated from thoracic ganglia, has the sequence Xaa-Arg-Pro-Tyr-Ser-Phe-Gly-Leu-NH2, where Xaa is either Asp or Asn. This peptide, with a serine substitution for glycine at position 5, has a C-terminal pentapeptide sequence identical to that of allatostatins 3 and 4 of D. punctata. Callatostatin 5, with the sequence Gly-Pro-Pro-Tyr-Asp-Phe-Gly-Met-NH2, was identified from whole flies. All five peptides inhibit juvenile hormone production by the corpora allata of D. punctata in vitro. Callatostatin 5 was the most potent allatostatin so far tested in this species, with maximum inhibition occurring at 1 nM. In contrast, none of the callatostatins or the allatostatins showed allatostatic activity in mature female C. vomitoria when tested at concentrations of 100 to 0.1 microM. In accordance with these results, immunoreactivity to an antiserum directed against the common C terminus of callatostatin 1 and allatostatin 1 was observed in the corpora allata of D. punctata but not in the corpus allatum of C. vomitoria, despite its presence in neurons of the brain. Neurons in the thoracic ganglion of C. vomitoria that are immunoreactive against this antiserum project to the hindgut, rectum, rectal papillae, and oviduct, suggestive of a function different from that of a true allatostatin.     

Characterization and cloning of tripeptidyl peptidase II from the fruit fly, Drosophila melanogaster

We describe the characterization, cloning, and genetic analysis of tripeptidyl peptidase II (TPP II) from Drosophila melanogaster. Mammalian TPP II removes N-terminal tripeptides, has wide distribution, and has been identified as the cholecystokinin-degrading peptidase in rat brain. Size exclusion and ion exchange chromatography produced a 70-fold purification of dTPP II activity from Drosophila tissue extracts. The substrate specificity and the inhibitor sensitivity of dTPP II is comparable to that of the human enzyme. In particular, dTPP II is sensitive to butabindide, a specific inhibitor of the rat cholecystokinin-inactivating activity. We isolated a 4309-base pair dTPP II cDNA which predicts a 1354-amino acid protein. The deduced human and Drosophila TPP II proteins display 38% overall identity. The catalytic triad, its spacing, and the sequences that surround it are highly conserved; the C-terminal end of dTPP II contains a 100-amino acid insert not found in the mammalian proteins. Recombinant dTPP II displays the predicted activity following expression in HEK cells. TPP II maps to cytological position 49F4-7; animals deficient for this interval show reduced TPP II activity.     

Intracellular responses of the rat cochlear nucleus to sound and its role in temporal coding

The anteroventral cochlear nucleus (AVCN), the first centre of the central auditory pathway, contains globular bushy cells, which are unique in their ability to produce fast excitatory post-synaptic potentials (EPSPs). Using in vivo intracellular recordings in the rat AVCN we examined these fast EPSPs in relation to temporal coding. At frequencies up to 2.5 kHz, EPSPs were evoked on successive sine waves of the stimulus with EPSP summation limited. This one-to-one relationship between the EPSPs and the sound wave period was present at higher frequencies and over a greater intensity range than for action potentials. These results suggest that temporal coding is possible in globular bushy neurones by their ability to extract temporal information through fast processing of convergent presynaptic input.     

Adrenergic responses in silent and putative inhibitory pacemaker-like neurons of the rat rostral ventrolateral medulla in vitro

Noradrenaline and adrenergic agonists were tested on pacemaker-like and silent neurons of the rat rostral ventrolateral medulla using intracellular recording in coronal brainstem slices as well as in punches containing only the rostral ventrolateral medullary region. Noradrenaline (1-100 microM) depolarized or increased the frequency of discharge of all cells tested in a dose-dependent manner. The noradrenaline-induced depolarization was associated with an apparent increase in cell input resistance at low concentrations and a decrease or no significant change at higher concentrations. Moreover, it was voltage dependent and its amplitude decreased with membrane potential hyperpolarization. Noradrenaline caused a dose-related increase in the frequency and amplitude of spontaneous inhibitory postsynaptic potentials. The alpha 1-adrenoceptor antagonist prazosin (0.5 microM) abolished the noradrenaline depolarizing response as well as-the noradrenaline-evoked increase in synaptic activity and unmasked an underlying noradrenaline dose-dependent hyperpolarizing response associated with a decrease in cell input resistance and sensitive to the alpha 2-adrenoceptor/antagonist yohimbine (0.5 microM). The alpha 1-adrenoceptor agonist phenylephrine (10 microM) mimicked the noradrenaline depolarizing response associated with an increase in membrane resistance as well as the noradrenaline-induced increase in synaptic activity. The alpha 2-adrenoceptor agonists UK-14,304 (1-3 microM) and clonidine (10-30 microM) produced only a small hyperpolarizing response, whereas the beta-adrenoceptor agonist isoproterenol (10-30 microM) had no effect. Baseline spontaneous postsynaptic potentials were abolished by strychnine (1 microM), bicuculline (30 microM) or both. However, only the strychnine-sensitive postsynaptic potentials had their frequency increased by noradrenaline or phenylephrine and they usually occurred with a regular pattern. Tetrodotoxin (1 microM) eliminated 80-95% of baseline spontaneous postsynaptic potentials and prevented the increase in synaptic activity evoked by noradrenaline and phenylephrine. Similar results were obtained in rostral ventrolateral medulla neurons impaled in both coronal slices and punches of the rostral ventrolateral medulla. It is concluded that noradrenaline could play an important inhibitory role in the rostral ventrolateral medulla via at least two mechanisms: an alpha 2-adrenoceptor-mediated hyperpolarization and an enhancement of inhibitory synaptic transmission through activation of alpha 1-adrenoceptors located on the somatic membrane of glycinergic interneurons. Some of these interneurons exhibit a regular discharge similar to the pacemaker-like neurons and might, at least in part, constitute a central inhibitory link in the baroreceptor-vasomotor reflex pathway.     

Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells

The observation that TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNF cytokine family, induces apoptosis in a number of different tumor cell types led us to compare the tumoricidal effects of TRAIL to those of other TNF family molecules on human melanoma cells. We found that a high proportion of the melanoma cell lines tested were killed by TRAIL, whereas all the melanoma lines were resistant to the other TNF family cytokines tested. TRAIL-induced death was characterized by caspase activation and cellular protein cleavage within minutes of TRAIL addition, and death could be completely inhibited by the caspase inhibitors Ile-Glu-Thr-Asp (IETD) and Val-Ala-Asp (VAD), indicating the presence of a TRAIL receptor signaling pathway similar to that identified for Fas and TNF receptors. Specific TRAIL receptor expression was determined by RT-PCR, and the presence of mRNA encoding the "protective" TRAIL receptors did not correspond to resistance or sensitivity to TRAIL-induced apoptosis. Addition of protein synthesis inhibitors to TRAIL-resistant melanomas rendered them sensitive to TRAIL, indicating that the presence or the absence of intracellular apoptosis inhibitors may mediate resistance or sensitivity to TRAIL-mediated apoptosis. Expression of one such inhibitor, FLICE-inhibitory protein (FLIP), was highest in the TRAIL-resistant melanomas, while being low or undetectable in the TRAIL-sensitive melanomas. Furthermore, addition of actinomycin D to TRAIL-resistant melanomas resulted in decreased intracellular concentrations of FLIP, which correlated with their acquisition of TRAIL sensitivity. Collectively, our results indicate that TRAIL-induced apoptosis occurs through a caspase signaling cascade and that resistance is controlled by intracellular regulators of apoptosis.