Synergistic interaction between the two mechanisms of action of tapentadol in analgesia.

The novel centrally acting analgesic tapentadol [(-)-(1R,2R)-3-(3-dimethylamino-1-ethyl-2-methyl-propyl)-phenol hydrochloride] combines two mechanisms of action, µ-opioid receptor (MOR) agonism and noradrenaline reuptake inhibition (NRI), in a single molecule. Pharmacological antagonism studies have demonstrated that both mechanisms of action contribute to the analgesic effects of tapentadol. This study was designed to investigate the nature of the interaction of the two mechanisms. Dose-response curves were generated in rats for tapentadol alone or in combination with the opioid antagonist naloxone or the α(2)-adrenoceptor antagonist yohimbine. Two different pain models were used: 1) low-intensity tail-flick and 2) spinal nerve ligation. In each model, we obtained dose-effect relations to reveal the effect of tapentadol based on MOR agonism, NRI, and unblocked tapentadol. Receptor fractional occupation was determined from tapentadol's brain concentration and its dissociation constant for each binding site. Tapentadol produced dose-dependent analgesic effects in both pain models, and its dose-effect curves were shifted to the right by both antagonists, thereby providing data to distinguish between MOR agonism and NRI. Both isobolographic analysis of occupation-effect data and a theoretically equivalent methodology determining interactions from the effect scale demonstrated very pronounced synergistic interaction between the two mechanisms of action of tapentadol. This may explain why tapentadol is only 2- to 3-fold less potent than morphine across a variety of preclinical pain models despite its 50-fold lower affinity for the MOR. This is probably the first demonstration of a synergistic interaction between the occupied receptors for a single compound with two mechanisms of action.

[Tapentadol: with two mechanisms of action in one molecule effective against nociceptive and neuropathic pain. Preclinical overview]. / Tapentadol: mit zwei Mechanismen in einem Molekül wirksam gegen nozizeptive und neuropathische Schmerzen. Präklinischer Überblick.

Tapentadol (3-[(1R, 2R)-3-(dimethylamino)-1-ethyl-2-methylpropyl] phenol) is a centrally acting analgesic of a new substance class for the treatment of severe nociceptive and neuropathic pain. Tapentadol combines µ-opioid receptor (MOR) agonism and noradrenaline reuptake inhibition (NRI) in one molecule. Because of the combined mechanisms of action tapentadol offers a broad therapeutic spectrum for nociceptive as well as neuropathic pain. In different animal models its high efficacy was shown in acute nociceptive, acute and chronic inflammatory as well as in chronic neuropathic pain. Using several preclinical approaches it was shown that the noradrenergic component of tapentadol interacts with the opioid component and that both synergistically contribute to the analgesic effect of the substance. In comparison to known drugs with only one of the two modes of action, tapentadol, despite its high potency, has an improved tolerability profile in the relevant animal models, particularly with regard to gastrointestinal and central side effects. Tapentadol acts directly without metabolic activation and without formation of analgesically relevant metabolites. In different interaction studies a low potential for interactions was shown, thus clinically relevant drug-drug interactions are unlikely. Overall, tapentadol provides a safe pharmacodynamic-pharmacokinetic profile.

Comparative effects of chlormadinone acetate and its 3alpha- and 3beta-hydroxy metabolites on progesterone, androgen and glucocorticoid receptors.

AIM/METHODS: In vitro binding tests to human receptors and in vivo functional activities in animals were used to compare the effects of the progestin chlormadinone acetate (CMA) and its 3alpha- and 3beta-hydroxy metabolites (3alpha-OH-CMA and 3beta-OH-CMA) on progesterone, androgen and glucocorticoid receptors. RESULTS: CMA, 3alpha-OH-CMA, 3beta-OH-CMA and the reference progestin R5020 bound to human progesterone receptor with Ki values of 2.5 nm, 13 nm, 6.0 nm and 4.3 nm, respectively. Binding affinities to the human androgen receptor were characterized by Ki values of 3.8 nM for CMA, 83 nM for 3alpha-OH-CMA, 20 nM for 3beta-OH-CMA and 2.9 nM for the reference androgen methyltrienolone. The Ki values for binding to the human glucocorticoid receptor were 16 nM for CMA, 69 nM for 3alpha-OH-CMA, 21 nM for 3beta-OH-CMA and 1.2 nM for the glucocorticoid dexamethasone. In the rabbit endometrial proliferation test CMA, 3alpha-OH-CMA and 3beta-OH-CMA (5 and 45 microg/kg p.o. for 5 days) had similar progestomimetic activities. CMA, 3alpha-OH-CMA and, to a lesser extent, 3beta-OH-CMA (4.64 and 21.5 mg/kg p.o. for 7 days) inhibited testosterone-stimulated growth of prostate and seminal vesicles in castrated rats showing antiandrogenic activities. Glucocorticoid properties were demonstrated for CMA and 3alpha-OH-CMA (21.5 and 100 mg/kg p.o. for 6 days) but not for 3beta-OH-CMA as reduction in thymus and adrenal gland weights in immature rats. CONCLUSION: Binding assays at human receptors showed similarly high affinities of CMA with the progesterone and androgen receptors and a 5 times lower affinity with the glucocorticoid receptor. At all receptor types, CMA had the highest, 3alpha-OH-CMA the lowest and 3beta-OH-CMA an intermediate affinity. Animal studies revealed progestomimetic and antiandrogenic activities of CMA, 3alpha-OH-CMA and 3beta-OH-CMA and glucocorticoid activities of CMA and 3alpha-OH-CMA.

Characterization of adenosine receptors in guinea-pig isolated left atria.

1. The effects of purinergic stimulation on action potential, force of contraction, 86Rb efflux and 45Ca uptake were investigated in guinea-pig left atria. 2. Adenosine exerted a negative inotropic effect which was antagonized by adenosine deaminase but enhanced by dipyridamole. 3. The negative inotropic effect of adenosine was mimicked by 5'-(N-ethyl)-carboxamido-adenosine (NECA) and the isomers of N6-(phenyl-isopropyl)-adenosine, R-PIA and S-PIA. NECA and R-PIA were about 100 times more potent than adenosine, whereas R-PIA was about 100 times more potent than S-PIA. 4. The inotropic effects of adenosine (in the presence of dipyridamole), NECA, R-PIA and S-PIA were competitively antagonized either by theophylline (pA2 about 4.5) or 8-phenyltheophylline (pA2 about 6.3). 5. NECA and R-PIA shortened the action potential duration and increased the rate constant of the efflux of 86Rb in a concentration-dependent manner with no differences in potency; the effects were competitively antagonized by 8-phenyltheophylline. 6. Barium ions reduced the efflux of 86Rb under control conditions and antagonized the increase induced by NECA and R-PIA. 7. NECA and R-PIA significantly reduced 45Ca uptake in beating preparations. 8. It is concluded that adenosine, NECA and R-PIA activate a common receptor population (P1 or A3) on the outside of the cell membrane of atrial heart muscle to increase the potassium conductance and to reduce the action potential and, thereby, calcium influx and force of contraction.

Different mechanisms of the inhibition of the transient outward current in rat ventricular myocytes.

The mechanism of drug-induced inhibition of the transient outward current, Ito, has been investigated in rat ventricular myocytes using the whole cell patch clamp technique. Ito was activated by 300 ms depolarizing voltage clamp steps in 10 mV increments from -50 mV up to +40 mV. At +40 mV, Ito peaked after about 3 ms, and the time course of inactivation was appropriately described by two time constants, tau fast = 17 ms and tau slow = 203 ms. Verapamil, quinidine sulfate and nifedipine preferentially depressed Ito at the end of the 300 ms depolarizing voltage clamp step; the inactivation of Ito was accelerated by all drugs, whereas peak Ito was less affected. The time course of drug action at +40 mV was calculated by the fractional changes of Ito. Verapamil, quinidine sulfate and nifedipine exerted a block of Ito increasing during the depolarizing voltage clamp step. The onset of block in response to verapamil, quinidine sulfate and nifedipine (30 mumol/each) was appropriately described by monoexponential functions with time constants tau on = 9.3, 1.7 and 1.1 ms, respectively. Relief from block by verapamil, quinidine sulfate and nifedipine at -50 mV was assessed by comparison of the recovery process of peak Ito from inactivation with or without drugs. tau off amounted to 695 ms in the case of quinidine sulfate; verapamil and nifedipine did not significantly affect the recovery process so that the determination of the time course of relief from block was not possible.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiologic and inotropic effects of alpha-adrenoceptor stimulation in human isolated atrial heart muscle.

The effects of alpha-adrenoceptor stimulation on force of contraction were investigated in human atrial heart muscle and compared with those of beta-adrenoceptor stimulation. The maximal positive inotropic effect produced by stimulation of alpha-adrenoceptors with phenylephrine (in the presence of atenolol 10 mumol/l) was significantly smaller than that seen in response to beta-adrenoceptor stimulation with isoprenaline. The maximal effect of phenylephrine (25% of the maximal effect of isoprenaline) required far higher concentrations (1 mmol/l) than isoprenaline (100 nmol/l); the EC50 values amounted to 33.1 mumol/l and 3.3 nmol/l, respectively. In the presence of the alpha-adrenoceptor blocking agent phentolamine (1 mumol/l), the concentration-response curve of phenylephrine was displaced to higher concentrations of the agonist; under these conditions, the EC50 value amounted to 52.5 mumol/l. The effects of the catecholamines noradrenaline and adrenaline on force of contraction remained unchanged in the presence of phentolamine (1 mumol/l) or prazosin (1 mumol/l). The positive inotropic effect of phenylephrine (1 mmol/l) was associated with a slight decrease in action potential duration; the effects on action potential were completely blocked in the presence of phentolamine (1 mumol/l). These findings support the view that selective stimulation of alpha-adrenoceptors may mediate a small but detectable positive inotropic effect in human atrial tissue under in vitro conditions. The requirement of high concentrations of alpha-adrenoceptor agonists and the lack of effects of the endogenous catecholamines adrenaline and noradrenaline on alpha-adrenoceptors (in concentrations which fully elicit the beta-adrenoceptors-mediated response) do not provide a basis for a functional role of alpha-adrenoceptor-mediated effects under in vivo conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenoceptor-mediated effects on calcium channel currents are antagonized by 5'-(N-ethyl)-carboxamido-adenosine in guinea-pig atrial cells.

In guinea-pig atrial myocytes, the effects of the adenosine analogue 5'-(N-ethyl)-carboxamido-adenosine (NECA) in the presence of isoprenaline (ISO) on Ca2+ channel activity were analyzed. Single Ca2+ channel currents were recorded from cell-attached patches by application of several hundred 100 ms depolarizing steps. Under control conditions, burstlike activity of channel openings during some depolarizing steps were followed by variably long periods of quiescence (blank sweeps). During superfusion with ISO (100 nmol/l), ensemble-averaged (mean) current was increased by about 150%. The underlying mechanism was found to be a significant increase in the channel availability, defined as the ratio of current-containing sweeps to the total number of sweeps. In addition, the ISO-induced reduction of blank sweeps was combined with slightly but not significantly higher values of the open probability in the current-containing sweeps. Open time and shut time histograms could be fitted by single and double exponential curves, respectively, which remained rather unaffected in the presence of ISO; accordingly, mean open time and mean shut time of the channel were not significantly changed by ISO. After the addition of NECA (1 mumol/l) in the presence of ISO, the ISO-induced increase in mean current was abolished. This effect of NECA on mean current was due to a reduction of the channel availability and a slight decrease in the open probability. The purinoceptor blocking agent 8-phenyltheophylline (10 mumol/l) antagonized the inhibitory action of NECA on the ISO-induced increase in Ca2+ channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)

On the mechanism of action of phenylephrine in rat atrial heart muscle.

Both in rat left atrial heart and in aortic smooth muscle preparations, phenylephrine (PE) caused a concentration-dependent increase in force of contraction (FC) in the presence of atenolol (10 mumol/l), which was antagonized by phentolamine, prazosin and WB 4101 in a competitive manner. The pA2 values of the antagonists in the cardiac tissue were 10-20fold lower than those in the rat thoracic aorta. In the spontaneously beating right atrium, PE exerted a positive chronotropic action, which was not significantly antagonized by phentolamine or prazosin. It is therefore assumed that the effects of phenylephrine in the left atrium and in the aorta are mediated by different subtypes of alpha 1-adrenoceptors, whereas the effects in the sino-atrial node are probably unrelated to alpha 1-adrenoceptors. To further elucidate the mechanisms of the positive inotropic effect of PE, action potential configuration and 45Ca2+ fluxes were monitored in the rat left atrium. The increase in FC by PE was associated with an increase in action potential duration (APD) and a reduction in resting membrane potential (RP). In the presence of (-)-devapamil (D888), the effects of PE on APD and RP persisted, whereas the increase in FC was antagonized in a non-competitive manner. Forskolin (300 nmol/l) enhanced the positive inotropic effect of PE. PE exerted a significant increase in 45CA2+ uptake in beating preparations, which was abolished in the presence of (-)D888 (1 mumol/l). In addition to the PE-induced increase in 45Ca2+ uptake, a decrease in 45Ca2+ efflux was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Contribution of both alpha- and beta-adrenoceptors to the inotropic effects of catecholamines in the rabbit heart.

The functional role of alpha-adrenoceptors was investigated in different parts of the rabbit heart. Phenylephrine (PE) caused a marked increase in force of contraction (Fc) and a prolongation of the action potential (AP) in preparations from the left atrium and the right ventricle. The response was less pronounced in the right atrium and in the left ventricle, whereas APs of spontaneously beating sinoatrial preparations remained completely unchanged. Phentolamine as well as the diesters phorbol 12,13 dibutyrate (PDBu) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) eliminated the effects of PE. The contribution of alpha-adrenoceptors to the effects of adrenaline (Adr) and noradrenaline (NA) on Fc was determined in preparations from the right ventricle. Phentolamine and the phorbol diesters reduced the effects of Adr and NA by about 30 to 60%; the remaining response was abolished by propranolol. It can be derived from our experiments that, in some parts of the rabbit heart, a considerable amount of the effects of Adr and NA is due to the stimulation of alpha-adrenoceptors. The present findings therefore support the view that, in the rabbit heart, the maximally effective drive of the heart requires the stimulation of both alpha- and beta-adrenoceptors. The inhibitory effects of phorbol diesters on the alpha-adrenoceptor-mediated response indicate that the activation of protein kinase C (PKC) specifically uncouples alpha-adrenoceptors from the effector system, whereas the response to beta-adrenoceptor stimulation remains unchanged.

Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle.

The effects of 5-hydroxytryptamine (5-HT) on force of contraction (FC), action potential (AP) and calcium current (ICa) were studied in human right atrial and left ventricular heart muscle. 5-HT exerted a concentration-dependent increase in FC in multicellular atrial preparations; the EC50 was approximately 3 x 10(-7) mol/l. Maximal increases in FC (252 +/- 58% of control values; mean +/- SEM, n = 6) were obtained at 5-HT 10(-5) mol/l. At this concentration, ICa was increased four- to sevenfold in enzymatically isolated atrial myocytes. In contrast, ventricular preparations did not respond to 5-HT; FC, AP and ICa remained unaffected. In the same preparations, FC was increased by isoprenaline three- to fourfold. These results confirm the observation that 5-HT induces a positive inotropic effect in the human atrium, possibly mediated by activation of the adenylyl cyclase - cyclic AMP system. Our study demonstrates, however, the complete lack of functional 5-HT receptors, with respect to changes in FC, in the human ventricle. Since the positive inotropic effect of 5-HT in the human heart is obviously restricted to the atrium, our findings question the concept of developing 5-HT receptor agonists for the treatment of heart failure.

L-type calcium channel activity in human atrial myocytes as influenced by 5-HT.

5-Hydroxytryptamine (10 mumol/l; 5-HT) exerted a positive inotropic effect associated with an increase in the Ca2+ current (ICa) in the human right atrium. For detailed analysis, L-type Ca2+ channel currents were recorded from cell-attached patches using 100 mmol/l Ba2+ as charge carrier. Ca2+ channel activity was identified, first, by burst-like inwardly directed currents and, second, by the appearance of long channel openings promoted by Bay K 8644 (1 mumol/l) upon repetitive depolarizations from -80 to 0 mV. The unitary conductance of the Ca2+ channel amounted to 25.8 pS. During superfusion with 5-HT, ensemble averaged (mean) current was enhanced by about 60%. The increase in mean current was brought about by an increase in the channel availability, defined as the ratio of sweeps containing Ca2+ channel activity to the total number of depolarizations. The open probability of a single Ca2+ channel within a sweep with channel activity, unitary conductance, mean open and mean shut times of the channel, however, remained unaffected during superfusion with 5-HT (n = 10). The 5-HT-induced increase in macroscopic ICa in the human atrium can therefore be explained by an enhanced availability of Ca2+ channels to open upon depolarization. The observed changes in gating properties of the human Ca2+ channel by 5-HT are very similar to those which are known from isoprenaline-induced cAMP-dependent phosphorylation of the Ca2+ channel protein in other tissues.

Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats.

Stimulation of alpha 1-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca2+ current (ICa) and K+ currents were comparatively studied in either tissue. In atrial preparations, phenylephrine 10 mumol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 mumol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 mumol/l. In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 mumol/l did not affect the magnitude of ICa in either cell type. Transient and steady state K+ outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes. It is concluded that the positive inotropic effect of alpha 1-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K+ currents.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of cytosolic free calcium concentration by alpha 1-adrenoceptors in rat atrial cells.

The effects of alpha 1-adrenoceptor stimulation by phenylephrine (PE) and beta-adrenoceptor stimulation by isoprenaline (ISO) on Ca2+ current (ICa) and free intracellular Ca2+ concentration ([Ca2+]i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of ICa, whereas large increases of peak ICa were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca2+]i and, initially, an increase in the height of peak [Ca2+]i transients. When the diastolic [Ca2+]i was increased to a greater extent, the amplitude of [Ca2+]i transients was decreased. Simultaneous measurements of [Ca2+]i and membrane potential showed that the increase in diastolic [Ca2+]i was associated with a depolarization of the membrane, and the greater amplitude of [Ca2+]i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca2+]i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca2+]i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca2+]i transients with only minor changes in diastolic [Ca2+]i. These results suggest that PE and ISO increase the amplitude of [Ca2+]i transients in rat atrium in different ways. The increase in [Ca2+]i transients in response to beta-adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca2+ channels causing a greater Ca2+ influx and a release of more Ca2+ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca2+]i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na(+)-Ca2+ exchange mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanical and electrophysiological effects of cromakalim on the human urinary bladder.

The effects of cromakalim on spontaneous and induced mechanical activity of human detrusor muscle were investigated in vitro. Cromakalim produces a concentration-related decrease of spontaneous as well as carbachol- and K(+)-evoked contractions. This is the first study to utilize the patch clamp technique to elucidate the mechanism of action of cromakalim on human detrusor cells. Cromakalim hyperpolarizes the detrusor cells by increasing the net outward current which is most likely carried by potassium ions. In the human urinary bladder, this effect is mediated by a glibenclamide-sensitive potassium channel, as glibenclamide is able to diminish the relaxant effect of cromakalim and to prevent the drug-induced hyperpolarization. Our results show that the effectiveness of cromakalim in relaxing the detrusor muscle is about an order of magnitude higher in the rat and guinea pig than in humans. It is therefore deemed unlikely that cromakalim or drugs with the same mode of action and similar tissue specificity will be of value in the clinical treatment of bladder instability and hyperreflexia. Moreover, it is suggested that when studies are undertaken to elucidate the effects and clinical potential of new drugs acting upon the bladder, human detrusor tissue should be used. The rat and guinea pig are unsuitable for this purpose.

Depolarization-induced influx of sodium in response to phenylephrine in rat atrial heart muscle.

1. The effects of alpha 1-adrenoceptor stimulation on transmembrane potential, currents and ion fluxes were investigated in multicellular preparations and/or single cells obtained from the left atrium of rat hearts. 2. In multicellular preparations, phenylephrine caused a concentration-dependent positive inotropic effect, an increase in action potential duration, and a decrease in resting potential; the effects were antagonized by phentolamine. 3. In the presence of phenylephrine (100 mumol/1), two levels of resting potential were observed when the preparations were, alternately, electrically stimulated or kept at rest (-74 +/- 1 mV during activity and -62 +/- 4 mV at rest; mean +/- S.E.M.; n = 9). 4. In resting preparations, the depolarization in response to phenylephrine was eliminated in low-Na+ solution (12 mmol/l) and antagonized by tetrodotoxin (10 mumol/l). 5. The phenylephrine-induced depolarization was also seen in nominally Ca(2+)-free solution and in the presence of (-)-devapamil (1 mumol/l). 6. The alkylating agent N-ethyl-maleimide (30 mumol/l) abolished the depolarizing effect of phenylephrine. 7. Phorbol 12,13-dibutyrate (10 mumol/l) also abolished the depolarizing effect of phenylephrine. 8. Phenylephrine caused a significant increase of 22Na+ uptake in resting preparations and of 45Ca2+ uptake in beating preparations. 9. The depolarizing effect of phenylephrine was also observed in single atrial myocytes. Steady-state membrane currents in response to 500 ms depolarizing and hyperpolarizing voltage clamp steps were decreased. The cross-over of I-V curves under control and test conditions was at about -70 mV. The effects of phenylephrine were antagonized in the presence of phentolamine. 10. After suppression of potassium currents by substitution of CsCl for internal and external KCl ([KCl]o), phenylephrine had no effect on membrane currents. 11. In conclusion, we presume the following sequence of events in response to phenylephrine in rat atrial heart muscle. First, the stimulation of alpha 1-adrenoceptors decreases the K+ conductance thereby producing a depolarization in the presence of an inward current. Second, the change of the membrane potential in the depolarizing direction induces a TTX-sensitive Na+ window current which further propels the depolarization. Third, the increase in Na+ influx may increase Ca2+ influx by activating the Na(+)-Ca2+ exchange in mechanism. The greater influx of Ca2+ may contribute to the positive inotropic effect in response to phenylephrine.

The antigen self-presentation function of the cytotoxic T-cell clone 10BK.1 depends on reciprocal peptide presentation.

Cells of the cytotoxic T-cell clone 10BK.1 proliferate in response to ovalbumin (OVA) not only in the presence, but also in the absence, of antigen-presenting cells. This response is the consequence of reciprocal antigen presentation by two 10BK.1 cells. At the single-cell level, we found antigen-induced changes in the cytosolic Ca2+ concentration ([Ca2+]i) only in 10BK.1 cells that had contact with another 10BK.1 cell. This finding suggests that a single cell is unable to present the antigen to itself, and that two 10BK.1 cells are necessary and adequate to present the antigen to each other. Our data indicate that 10BK.1 cells are not capable of processing the OVA molecule. The naturally processed peptide OVA257-264 is a very potent antigen for these cells. A corresponding peptide seems to be present not only in preparations of native OVA, but also in preparations of the commonly used peptide OVA258-276. This 19mer peptide does not bind with high affinity to the relevant Kb class I molecule.