Introduction

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In 1992, Raiteri and colleagues concluded that the presynaptic ₂-autoreceptors that modulate norepinephrine release in the human neocortex are distinct from _2B and _2C and are either _2A or _2D. This conclusion was mainly based on three findings: the potent release-inhibiting effect of oxymetazoline; the potent antagonism by yohimbine as opposed to the weak antagonism by prazosin and 2-{2-[4-(o-methoxyphenyl)piperazin-1-yl]ethyl}-4,4-dimethyl-1,3(2H,4H)-isoquinolinedione (ARC 239) of the release-inhibiting effect of clonidine; and the marked release-enhancing effect of yohimbine as opposed to the lack of a release-enhancing effect of prazosin and ARC 239 when these antagonists were given alone. All observations were in accord with the affinities of oxymetazoline, yohimbine, prazosin, and ARC 239 for _2A- and _2D-adrenoceptors but were not compatible with their affinities for _2B- and _2C-adrenoceptors (Raiteri et al., 1992).

It is now thought that the _2A- and _2D-adrenoceptors are species orthologs, of which only one occurs in a given species, and that humans possess the _2A version, whereas rodents possess the _2D version (see Bylund, 1995). Human neocortical ₂-autoreceptors therefore should be _2A. If so, they would obey the rule that the main mammalian ₂-autoreceptors belong to the _2A/D branch of the adrenoceptor tree (Trendelenburg et al., 1993, 1999; see Docherty, 1998). Recent evidence indicates, however, that noradrenergic neurons in addition may possess _2C-autoreceptors (Trendelenburg et al., 1997; see Docherty, 1998; Ho et al., 1998; Altman et al., 1999).

Because the identification of a receptor type by means of an agonist (oxymetazoline in the work of Raiteri et al., 1992) is ambiguous and because these authors used only three antagonists, we reinvestigated the subtype to which the ₂-autoreceptors belong in the human neocortex. For this purpose, we quantified the release-enhancing effect of nine antagonists, including prazosin and ARC 239. The concentration-response data were evaluated by fitting a logistic function, which yielded the maximal enhancement and the EC₅₀ of the antagonist. The effect of exogenous norepinephrine under autoinhibition-free as well as autoinhibition conditions was also studied. The evaluation of the norepinephrine concentration-response curves suggested proportionality between ₂-autoreceptor occupation and response and allowed the conversion of the EC₅₀ values of the antagonists into their dissociation constants, K_b values, at the autoreceptors. Thus, the subclassification of the ₂-autoreceptor in human neocortex was possible, based on functionally defined K_b values of antagonists in comparison with corresponding values from the literature. The conversion of EC₅₀ to K_b justified the use of the functional parameter K_b as an estimate of the antagonist dissociation constant. Thus, the bias or shift between EC₅₀ values and dissociation constants of binding experiments could be bridged.

	Materials and Methods

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Fresh neocortical tissue was obtained from patients during surgical access to subcortical tumors. The procedure was approved by the local Ethics Committee. The patients (n = 42) were of either sex and were between 21 and 86 years old. After premedication with midazolam or chlordiazepoxide, patients were anesthetized with thiopental, fentanyl, or flunitrazepam. Pancuronium was given for muscle relaxation. The tissue was immersed in ice-cold medium (see below) and processed immediately.

Cortical slices, 350 µm thick and perpendicular to the surface, were incubated with 0.1 µM ()-[³H]norepinephrine in 4 ml of medium for 45 min at 37°C and then superfused with [³H]norepinephrine-free medium at 0.4 ml/min. For electrical stimulation, rectangular pulses of 2-ms width and a voltage drop of 11 V across the electrodes of each superfusion chamber were used, yielding a current strength of approximately 76 mA (Stimulator I; Hugo Sachs Elektronik, Hugstetten, Germany). Four stimulation periods were applied (S₁ to S₄); they began after t = 75, 110, 145, and 180 min (t = 0 being the start of superfusion). To evoke release free of autoinhibition, each stimulation period consisted of two trains of 4 pulses/100 Hz, with a train interval of 2 min [pseudo-one-pulse conditions; Singer, 1988; Allgaier et al., 1995]. To evoke autoinhibited release, each stimulation period consisted of 90 pulses/3 Hz. Successive 5-min samples of the superfusate were collected from t = 60 min onward. Unlabeled norepinephrine (tested under pseudo-one-pulse conditions as well as at 90 pulses/3 Hz) or -adrenoceptor antagonists (tested at 90 pulses/3 Hz only) were added at increasing concentrations 15 min before S₂, S₃, and S₄. At the end of experiments, tissues were dissolved, and tritium was determined in superfusate samples and tissues.

The medium used for tissue collection, incubation, and superfusion contained 118 mM NaCl, 1.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 25 mM NaHCO₃, 1.2 mM KH₂PO₄, 11 mM glucose, and 0.57 mM ascorbic acid. It was saturated with a mixture of 95% O₂ and 5% CO₂. The superfusion medium also contained 1 µM desipramine or, in experiments with >1 µM exogenous norepinephrine, 10 µM desipramine plus 10 µM (+)-oxaprotiline.

The outflow of tritium was calculated as a fraction of the tritium content of the slice at the onset of the respective collection period (fractional rate; min¹). The overflow elicited by electrical stimulation was calculated as the difference: total tritium outflow during the collection period in which stimulation was applied and during the two collection periods thereafter minus estimated basal outflow; basal outflow was assumed to decline linearly from the collection period before to the collection period 10 to 15 min after onset of stimulation. The evoked overflow was then expressed as a percentage of the tritium content of the slice at the time of stimulation. For further evaluation, ratios were calculated for the overflow evoked by S₂, S₃, and S₄ and the overflow evoked by S₁. Moreover, effects of exogenous norepinephrine and of the antagonists were calculated for each single slice as a percentage of control, using the corresponding mean average control S₂/S₁, S₃/S₁, and S₄/S₁ ratios (solvent-treated slices, no agonist, no antagonist) as the reference. Drug effects on the basal efflux of tritium were evaluated similarly, based on values immediately before stimulation periods (b₁, etc.).

Concentration-response data were evaluated as follows. In the case of the inhibitory effect of exogenous norepinephrine under autoinhibition-free conditions, a logistic function was fitted to the "percentage of control" data to yield the maximal effect of norepinephrine I_{max observed}, its IC₅₀, and the slope parameter c (eq. 7 of Feuerstein and Limberger, 1999). In the case of the effect of exogenous norepinephrine under autoinhibition conditions, a special function was fitted to the data that describes the combined effects of exogenous and endogenous norepinephrine, assumes the proportionality of receptor occupation and the effect of norepinephrine, and yields the maximal obtainable effect of norepinephrine under autoinhibition-free conditions I_{max derived}, the dissociation constant K_d of the norepinephrine-autoreceptor complex, and the concentration of released transmitter norepinephrine at the autoreceptors in the absence of exogenous norepinephrine [NE_tr] (biophase concentration; eq. 14 of Feuerstein and Limberger, 1999). In the case of the facilitatory effect of the antagonists, we fitted a logistic function to the percentage of control data to obtain the E_max of the antagonist and its EC₅₀ [eq. 7 of Feuerstein and Limberger, 1999, adapted for enhancement instead of inhibition, i.e., S_x/S₁ = 1 + E_max × 10^p[B]/(10^pEC50 + 10^p[B]), where p[B] is the negative logarithm of the applied antagonist concentration and pEC₅₀ is the negative logarithm of EC₅₀]. The conversion of antagonist EC₅₀ to antagonist dissociation constant K_b was then based on the K_d and I_{max derived} of norepinephrine, determined independently as described above.

Results are given as arithmetic means or estimates with 95% confidence intervals (CI₉₅) in parentheses to indicate statistical probability (Altman, 1991). n is the number of brain slices.

Purchased drugs were ()-[ring-2,5,6-³H]norepinephrine, specific activity 40.5 Ci/mmol (DuPont, Dreieich, Germany); ()-norepinephrine hydrogen tartrate, desipramine HCl, corynanthine HCl, (±)-idazoxan HCl (Sigma, Deisenhofen, Germany); (+)-oxaprotiline HCl, phentolamine HCl (Novartis, Basel, Switzerland); spiroxatrine, (±)-2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane HCl (WB4101) (Biotrend, Köln, Germany); rauwolscine HCl (Roth, Karlsruhe, Germany); prazosin HCl (Pfizer, Karlsruhe); 6-chloro-9-((3-methyl-2-butenyl)oxy)-3-methyl-1H-2,3,4,5-tetrahydro-3-benzazepine maleate (SKF104078) (Smith Kline Beecham, Palo Alto, CA); and ARC 239 (Thomae, Biberach, Germany). Drugs were dissolved in distilled water, except for WB4101 (1 mM HCl) and spiroxatrine (10 mM HCl).

	Results

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The basal outflow of tritium (b₁) from slices superfused with medium containing 1 µM desipramine was 0.0034 (0.0033, 0.0035) min¹ (n = 389), the overflow of tritium elicited by two trains of 4 pulses/100 Hz (S₁) averaged 0.69 (0.64, 0.74) % of tissue tritium (n = 132), and the overflow elicited by 90 pulses/3 Hz (S₁) was 3.07 (2.92, 3.23) % of tissue tritium (n = 257). When the medium contained 10 µM desipramine and 10 µM (+)-oxaprotiline, the basal outflow of tritium as well as the evoked overflow were similar; 10 µM desipramine and 10 µM (+)-oxaprotiline were used in experiments with high concentrations of unlabeled norepinephrine (>1 µM) to ensure blockade of the uptake of the unlabeled amine.

Stimulation by two trains of 4 pulses/100 Hz led to autoinhibition-free release of [³H]norepinephrine, as shown previously (Allgaier et al., 1995) and confirmed here by the lack of an overflow-enhancing effect of 1 µM rauwolscine, added after S₁ (n = 9 versus 6 controls). Stimulation by 90 pulses/3 Hz, in contrast, led to autoinhibited release, as shown by the effects of the antagonists (see below).

Effect of Exogenous Norepinephrine. Unlabeled norepinephrine, when added before S₂, S₃, and S₄ at increasing concentrations, progressively reduced the electrically evoked overflow of tritium, both under autoinhibition-free conditions (Fig. 1A) and under conditions in which autoinhibition developed (Fig. 1B). Norepinephrine did not change the basal efflux of tritium. The concentration-response curve in Fig. 1A was obtained by logistic curve fitting (eq. 7, Feuerstein and Limberger, 1999). The curve in Fig. 1B was obtained by fitting a function that takes the effect of transmitter norepinephrine into consideration and is based on the assumption of proportionality between receptor occupation by norepinephrine and effect (eq. 14, Feuerstein and Limberger, 1999). The two concentration-response curves obviously differ: under autoinhibition conditions (Fig. 1B), the curve is shifted to the right and the maximal observed degree of inhibition is smaller. The parameters estimated from the curves are shown in Table 1. The parameters obtained under autoinhibition-free conditions by logistic curve fitting can be read easily from Fig. 1A: I_{max observed} is the asymptotic maximal inhibition, which the experimental curve approaches at high concentrations of exogenous norepinephrine, and pIC₅₀ is the abscissa of the point of inflection (Fig. 1A). The parameters derived from the data obtained under autoinhibition conditions, in contrast, cannot be read immediately from inspection of Fig. 1B: I_{max derived} is not the asymptotic maximal inhibition that the experimental curve approaches at high concentrations of exogenous norepinephrine and is superimposed on a background of ongoing autoinhibition, but it is the maximal inhibition obtainable with norepinephrine, whether released or exogenous, against an autoinhibition-free background. Moreover, K_d is not the concentration of norepinephrine causing 50% of the asymptotic maximal inhibition of the experimental curve but is the dissociation constant of the norepinephrine-autoreceptor complex, calculated on the basis of proportionality between receptor occupation and effect, as mentioned.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of unlabeled norepinephrine (NE) on evoked overflow of tritium. Cortical slices of human neocortex were preincubated with [3H]norepinephrine and then superfused and stimulated electrically (S1 to S4) either under autoinhibition-free conditions (A; two trains of 4 pulses/100 Hz, train interval 2 min) or under autoinhibition conditions (B; 90 pulses/3 Hz). Unlabeled norepinephrine was added at increasing concentrations before S2, S3, and S4. Ordinates, evoked overflow of tritium as a percentage of control, based on Sx/S1 values. The curve in A was obtained by fitting eq. 7, and the curve in B by fitting eq. 14 of Feuerstein and Limberger (1999). Each point represents a single brain slice.

Effect of -Adrenoceptor Antagonists. As shown in Fig. 2, all antagonists, when added before S₂, S₃, and S₄ at increasing concentrations, increased the overflow of tritium evoked by 90 pulses/3 Hz, indicating autoinhibition of transmitter release. For each antagonist, the data were evaluated by logistic curve fitting [E/E_max = 10^L/(10^pEC50 + 10^L), where L is the used log concentrations (M) of the antagonists]. The individual E = S_x/S₁ values scattered considerably, and clear maxima were not reached for several antagonists (Fig. 2). For these reasons, probably, the iterative calculations used to estimate the confidence intervals of the logistic parameters E_max and pEC₅₀ and an additional slope factor c did not converge when all three were left unconstrained. The slope factor, c, therefore, was constrained to 1 (see function E/E_max above). The parameter estimates are summarized in Table 2.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of -adrenoceptor antagonists on evoked overflow of tritium. Cortical slices of human neocortex were preincubated with [3H]norepinephrine and then superfused and stimulated electrically (S1 to S4) under autoinhibition conditions (90 pulses/3 Hz). Antagonists were added at increasing concentrations before S2, S3, and S4. Ordinates, evoked overflow of tritium as a percentage of control, based on Sx/S1 values. Curves were obtained by fitting eq. 7 of Feuerstein and Limberger (1999), constraining the slope parameter c to 1. Up arrows indicate that the corresponding numerical values given were outside the range of the y axis.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Diagram on the steps of the conversion EC50  Kb. NE, norepinephrine. The parameters obtained from Fig. 1A, pIC50, Imax observed, and c, yielded the concentration-inhibition sigmoid drawn (step 1). This graph allowed us to estimate the extent of inhibition at the abscissa point p[NEtr], i.e., the endogenous tone during S1 (step 2), which corresponded to an autoinhibition of 1  0.42 = 0.58 in the Sx/S1 paradigm (step 3). Half of this inhibition, a disinhibition by 50%, was 0.58/2 = 0.29, equivalent to a Sx/S1 ratio of 1  0.29 = 0.71. [NEtr] increased according to the increase in the Sx/S1 ratio (step 4). The equation at step 5 corresponds to eq. 7 of Feuerstein and Limberger (1999) with c = 1, including all estimated values and the antagonist concentration [B] = EC50-corr at half-maximum disinhibition. Step 6 yielded the solution of the equation: Kb.

	Discussion

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The subclassification of ₂-autoreceptors in human neocortex was achieved by using calculated dissociation constants, K_b values, of antagonist-autoreceptor complexes of nine -adrenoceptor antagonists in comparison to binding or functional data on these antagonists from the literature. The antagonist K_b values were obtained from their concentrations causing half-maximal disinhibition, EC₅₀ values. The following consideration was the rationale of this conversion, EC₅₀  K_b.

Usually the evaluation of the disinhibition of release to assess the affinity of release-enhancing antagonists is limited to the calculation of antagonist EC₅₀ values that are not K_b values (e.g., Limberger et al., 1995a,b). This restriction can be surmounted by the knowledge of the relationship between EC₅₀ and K_b. We first analyzed the interplay of exogenous and endogenous norepinephrine at the autoreceptors, using a previously developed model. The model assumes proportionality between receptor occupation and effect of norepinephrine or, in other words, assumes that the K_d of norepinephrine equals its concentration causing half-maximal inhibition, IC₅₀, of [³H]norepinephrine release under autoinhibition-free conditions. It should be noted that the IC₅₀ of exogenous norepinephrine under autoinhibition-free conditions, 10^8.07 M, in fact was almost identical to the calculated K_d, 10^7.99 M (Table 1). Thus, the present experiments have for the first time established the K_d of norepinephrine at a central human ₂-autoreceptor in functional experiments. The above-mentioned assumption of proportionality as a prerequisite for IC₅₀ = K_d is supported by the estimate near unity of the slope factor, c (0.98, Table 1). Because of the limited number of data points of Fig. 1A and the small S₁ values (0.69% of tissue tritium) that increased the variation in the S₂/S₁ ratio, the CI₉₅ of c, however, was large (0.65, 1.75). This precludes a low error probability of the statement c = 1 (see Agneter et al., 1997; Feuerstein and Limberger, 1999). Accordingly, the statement IC₅₀ = K_d would also have a rather large error probability if it was only based on this large CI₉₅ of c. We tried, therefore, to increase the specificity of the estimate of c by reassessing this value from the data of Fig. 1B. In other words, the "logistic components" of eq. 14 of Feuerstein and Limberger (1999) were endowed with a slope factor c, and this amended function was then refitted to the data of Fig. 2B with fixed values for pK_d = 7.99 and I_{max derived} = 0.83 (Table 1), i.e., with a reduction of the number of parameters to reach convergence. This seemed reasonable because the maximum ₂-autoreceptor-mediated effect had to be the same in the presence and in the absence of autoinhibition and because the pK_d (pIC₅₀ of Fig. 1B) was nearly identical to the pIC₅₀ of Fig. 1A. The refit yielded a value for p[NE_tr] corresponding to that of Table 1 (not shown) and an additional c of 1.03 (0.64, 1.42). Now two similar estimates for c were available, and their mean with deviation could be calculated (using the approximate standard errors of c, 0.17 and 0.18): 1.01 (0.77, 1.24). Thus the CI₉₅ of this mean c became considerably smaller, which improved our evidence for assuming c = 1 or IC₅₀ = K_d.

The dissociation constants K_b of the antagonists were calculated by the use of their EC₅₀ values, the K_d (=IC₅₀) of norepinephrine, I_{max observed} obtained under autoinhibition-free conditions, and the calculated endogenous concentration [NE_tr]. In addition, the observed mean of the maximum disinhibition by the antagonists, 60% (Table 2), was considered as follows. A theoretical maximum disinhibition, S_xmax/S₁, can be calculated on the basis of the values of Table 2. S_xmax is the stimulation-induced transmitter release that is not inhibited by the endogenous agonist [NE_tr], or, in other words, S_xmax is the stimulation-induced transmitter release when the autoreceptor is completely blocked (when the concentration of the antagonist [B]  ). At S₁, however, the stimulation-induced transmitter release is diminished to S₁ = 1  I_{max observed} × 10^pNEtr/(10^pKd + 10^pNEtr) [compare eq. 9 of Feuerstein and Limberger (1999)]. Thus S_xmax/S₁ = 1/(1  0.79 × 10^7.61/(10^8.07 + 10^7.61)) = 2.39. The theoretically expected value of 2.39, or an increase by 139%, is at variance with the observed mean of maximum disinhibition, which was 1.60, or an increase by 60% at the highest antagonist concentrations used (1-10 µM). This discrepancy may be due to the following condition. At the highest antagonist concentrations (up to 1000-fold of the K_b values) additional, nonspecific effects of the antagonists, not related to the ₂-autoreceptor under investigation, must be taken into account. These nonspecific effects are probably inhibitory, not stimulatory, in nature, i.e., they may diminish the increase in the evoked [³H]norepinephrine release because action potential-evoked, exocytotic release is a highly specific phenomenon that is dependent on the integrity of the neuronal environment. Decreasing nonspecific effects are much more likely than increasing nonspecific effects. As one example of perturbations by high antagonist concentrations of the release process local anesthetic inhibitory effects of yohimbine and rauwolscine at higher concentrations are well known (e.g., Goodall et al., 1984). Therefore, depressant, not stimulatory, effects of the antagonists at concentrations that are much higher than their K_b values may be assumed, and the evaluation of their concentration-disinhibition curves may be amended as follows. The depressions of the concentration-disinhibition curves of the antagonists at their highest concentrations, but not at rather low concentrations specific for -adrenoceptors, correspond to the condition of an uncompetitive, use-dependent antagonism, as opposed to a noncompetitive antagonism (Segel, 1975; Jackisch et al., 1994). If fitted with the usual logistic function, e.g., eq. 5 of Feuerstein and Limberger (1999), an apparent EC₅₀ is obtained that is too low. To obtain a real EC₅₀, E/E_max = 10^L/(10^pEC50 + 10^L × Depr-E_max) should be used, where Depr-E_max is the relative extent to which the uncompetitive mechanism depresses E_max, instead of E/E_max = 10^L/(10^pEC50 + 10^L). In our case, Depr-E_max is 2.39/1.60 = 1.49. With respect to the quantitatively dissimilar depressions by the nine antagonists, note that most of the CI₉₅ values of the E_max values of Table 2 overlap, suggesting a roughly similar depression of the theoretical maximum disinhibition. Therefore, 1.49 may be roughly the factor by which the too low EC₅₀ obtained with E/E_max = 10^L/(10^pEC50 + 10^L) may be corrected to get a more realistic EC₅₀, according to E/E_max = 10^L/(10^pEC50 + 10^L × Depr-E_max) (Jackisch et al., 1994). This correction yields the pEC_50-corr values of Table 2.

When the values pIC₅₀, p[NE_tr], I_{max observed}, EC_50-corr are introduced into eq. 7 of Feuerstein and Limberger (1999), step 5 in Fig. 3, (1 + [EC_50-corr]/K_b) = 8.37 or pK_b = pEC_50-corr + 0.87 is obtained. The corresponding K_b values for the antagonists represent the first dissociation constants of antagonists at human cerebral autoreceptors obtained in functional experiments (Table 2).

To subclassify the ₂-autoreceptors, the autoreceptor pK_b values were compared with dissociation constants at known subtypes by means of a correlation analysis (Table 3), as has become usual in the literature, e.g., Bylund et al. (1992). Note that use of the pEC₅₀ values in the correlation analyses would have sufficed for the identification of the ₂-autoreceptor subtype because the subsequent transformation to pK_b values has no effect on the correlation coefficients. However, apart from obtaining accurate pK_b values, we wanted to demonstrate that the potency of an antagonist in disrupting the autoinhibitory circuit of transmitter release is a direct measure of its dissociation constant at the receptor to be blocked.

View this table: [in this window] [in a new window]   TABLE 3 Correlation between pKb values of antagonists at the presynaptic 2-autoreceptors in human neocortex and pKd values at previously subclassified 2 sites Shown are correlation coefficients (r), P values indicating the significance of the difference between r and 0, and slopes of the regression "pKd at previously subclassified 2 sites" on "pKb at 2-autoreceptors in human neocortex." pKb values at 2-autoreceptors in human neocortex are from Table 2. pKd values at previously subclassified 2 sites are from the references quoted.

The known subtypes of the literature were, first, prototypical native ₂ radioligand binding sites (Table 3A); second, radioligand binding sites in COS cells transfected with ₂-adrenoceptor genes (Table 3B); and third, previously subclassified ₂-autoreceptors (Table 3C). Table 3 shows that the dissociation constants of the antagonists at the human neocortical autoreceptors correlate significantly and without exception with their dissociation constants at both _2A and _2D binding sites or receptors; correlations with _2B and _2C binding sites or receptors are not significant (exception: the _2C binding sites in opossum kidney cells; Table 3A). Moreover, Table 3 shows that the coefficients for the correlation with _2A are generally higher than for the correlation with _2D, the error probability is generally lower in the former than in the latter case, and the slopes of the regression lines for _2A are generally closer to unity. We conclude that the ₂-autoreceptors in human brain cortex are _2A.

Presynaptic ₂-autoreceptors have also been subclassified in the human saphenous vein, kidney, and heart. In the saphenous vein, the receptors were suggested to be _2A (Molderings and Göthert, 1995), whereas in the kidney and heart they were initially classified as _2C (Trendelenburg et al., 1994; Rump et al., 1995). A reinvestigation of the kidney receptors, however, also yielded an _2A diagnosis (Trendelenburg et al., 1997). Overall, _2A (i.e., genetically _2A/D) autoreceptors seem to predominate in humans, as they do in various animal species (see the Introduction).

In summary, this paper shows that it is possible to analyze quantitatively the autoinhibitory circuit of [³H]norepinephrine release in human neocortex tissue, i.e., to estimate the biophase concentration of the transmitter in relation to its K_d, and to calculate true, unbiased dissociation constants of antagonists by evaluation of their disinhibition of [³H]norepinephrine release. The functionally obtained pK_b values at the presynaptic ₂-autoreceptors in human brain cortex correlated highly with pK_b values at previously subclassified _2A sites, but did not correlate significantly or correlated much less well with pK_b values at _2B, _2C, and _2D sites. It is concluded that the ₂-autoreceptors in human neocortex are _2A.