Therapeutic use of release-modifying drugs.

Presynaptic inhibitory or facilitatory autoreceptors are targets for the endogenous neurotransmitter of the respective neuron, and also for exogenous agonists, partial agonists and antagonists which can produce pharmacological actions through changes in transmitter release. In addition, presynaptic inhibitory or facilitatory heteroreceptors can also be acted upon by exogenous agonists, partial agonists or antagonists to induce changes in transmitter release with useful therapeutic effects. This article summarizes drugs that are known or likely to produce their therapeutic effects through presynaptic modulation of neurotransmitter release. Included are drugs acting on alpha and beta adrenoceptors, dopamine receptors, angiotensin, opioid, cannabinoid, and nicotinic acetylcholine receptors. Also discussed are changes in presynaptic receptor mechanisms produced by drugs that inhibit transmitter re-uptake.

History and nomenclature of alpha1-adrenoceptors.

Until 1974 it was widely accepted that alppha-adrenoceptors represented a homogeneous population of receptors. Following the discovery of presynaptic, release-modulating receptors and based on differences in potency for the alpha-adrenoceptor antagonist phenoxybenzamine, it was proposed in 1974 that alpha-adrenoceptors should be subdivided in alpha1-and alpha2-subtypes. The concept of subtypes of the alpha1-adrenoceptor was first suggested in the mid 1980s on the basis of the different affinities for the agonist, oxymetazoline, and the antagonists, WB4101 and phentolamine on certain 1-adrenoceptor mediated pharmacological preparations. Subsequent characterization of the alpha1-adrenoceptor using radioligand binding and functional studies has let to the identification of the three native prazosin-high-affinity alpha1-adrenoceptor subtypes designated alpha1A, alpha1B, and alpha1D, corresponding to the three alpha1-adrenoceptor subtypes (alpha1a, alpha1b, and alpha1d) characterized by molecular cloning techniques. Studies concerning the distribution of alpha1-adrenoceptors in the human prostate tissue have shown that the predominant cloned alpha1a-adrenoceptor subtype characterized by RNAase protection assays corresponds to the alpha1A-subtype. Both obstruction and lower urinary tract symptoms (LUTS) associated with benign prostatic hyperplasia (BPH) are enhanced by noradrenergic activation of stromal alpha1-adrenoceptors in prostate. Therefore, the prostatic alpha1-adrenoceptors have become an important target for the pharmacotherapeutic treatment of BPH. In this context, Alfuzosin was the first uroselective alpha1-adrenoceptor antagonist to be evaluated in the treatment of BPH and was subsequently marketed, initially in France, in 1987. This drug has since become the standard alpha1-adrenoceptor blocker in the treatment of BPH and is widely marketed in Europe. Many of the alpha1-adrenoceptor antagonists currently prescribed in the treatment of BPH do not exhibit in vitro selectivity between alpha1A, alpha1B, and alpha1D-subtypes and yet they have good clinical tolerance in terms of low incidence of cardiovascular effects. One possibility to explain these findings is that another alpha1-adrenoceptor subtype could be implicated in human prostatic smooth muscle contraction. There is some evidence that an alpha1-adrenoceptor subtype with lower affinity for prazosin designed 1L, which has not been cloned yet, may be the predominant alpha1-subtype involved in the contractile response of the human prostatic smooth muscle to noradrenaline.

Dysthymia: clinical picture, extent of overlap with chronic fatigue syndrome, neuropharmacological considerations, and new therapeutic vistas.

Dysthymia, as defined in the American Psychiatric Association and International Classification of Mental Disorders, refers to a prevalent form of subthreshold depressive pathology with gloominess, anhedonia, low drive and energy, low self-esteem and pessimistic outlook. Although comorbidity with panic, social phobic, and alcohol use disorders has been described, the most significant association is with major depressive episodes. Family history is loaded with affective, including bipolar, disorders. The latter finding explains why dysthymia, especially when onset is in childhood, can lead to hypomanic switches, both spontaneously and upon pharmacologic challenge in as many as 30%. Indeed, antidepressants from different classes -tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), reversible inhibitors of monoamine oxidase A (RIMAs), selective serotonin-reuptake inhibitors (SSRIs) and, more recently, amisulpride, and spanning noradrenergic, serotonergic as well as dopaminergic mechanisms of action - have been shown to be effective against dysthymia in an average of 65% of cases. This is a promising development because social and characterologic disturbances so pervasive in dysthymia often, though not always, recede with continued pharmacotherapy beyond acute treatment. Despite symptomatic overlap of dysthymia with chronic fatigue syndrome - especially with respect to the cluster of symptoms consisting of low drive, lethargy, lassitude and poor concentration - neither the psychopathologic status, nor the pharmacologic response profile of the latter syndrome is presently understood. Chronic fatigue today is where dysthymia was two decades ago. We submit that the basic science - clinical paradigm that has proven so successful in dysthymia could, before too long, crack down the conundrum of chronic fatigue as well. At a more practical level, we raise the possibility that a subgroup within the chronic fatigue group represents a variant of dysthymia.

Development of an inducible NMDA receptor stable cell line with an intracellular Ca2+ reporter.

Cytotoxicity associated with NMDA receptor activation has impeded the establishment of cell lines expressing recombinant subtypes of this ligand-gated ion channel class. To circumvent this toxicity, we describe in this report the use of a potent inducible promoter in the construction of a cell line stably expressing the NR1a/NR2A subtype of the NMDA receptor. Western blot analysis using subunit selective antibodies revealed that NR2A subunits were constitutively expressed in this cell line, whereas expression of NR1a subunits was tightly regulated by tetracycline. Upon tetracycline removal, electrophysiological recordings using the patch clamp technique indicated the expression of functional receptors with biophysical and pharmacological properties corresponding to those expected of the NR1a/NR2A subtype. In addition, we utilized this cell line with the recombinant membrane targeted Ca2+ reporter, aequorin, in a functional assay of NMDA receptor activation. An evaluation of the coupling efficiency of NMDA receptor activation and aequorin response, as well as the pharmacological profile of this assay, illustrates the suitability of this cell line and the Ca2+ reporter assay to functionally identify novel NMDA receptor antagonists.

Nomenclature and state of the art on alpha1-adrenoceptors.

The concept of alpha1-adrenoceptor subtypes was first suggested in the mid 1980s on the basis of the different affinities of certain alpha1-adrenoceptor preparations for the alpha-adrenoceptor agonist, oxymetazoline, and the antagonists, WB4101 and phentolamine. Subsequent characterization of alpha1-adrenoceptors using radioligand binding and functional studies has led to the identification of three native prazosin high-affinity alpha1-adrenoceptor subtypes designated alpha1A, alpha1B and alpha1D, corresponding to the three alpha1-adrenoceptor subtypes (alpha1a, alpha1b and alpha1d) isolated by molecular cloning techniques. Since each of these three subtypes exhibits similar affinity for the selective alpha1-adrenoceptor antagonist, prazosin, [3H]prazosin can be used as a convenient probe to evaluate the interaction of compounds with these adrenoceptor subtypes. Considerable clinical experience over the last few years has provided convincing evidence to support the effectiveness of alpha1-adrenoceptor blockade in the treatment of bladder obstruction due to benign prostatic hyperplasia (BPH). The distribution of alpha1-adrenoceptors in the human prostate tissue has shown that the predominant cloned alpha1-adrenoceptor subtype characterized by RNAase protection assays corresponds to the alpha1a-subtype, formerly classified as alpha1c. Many of the alpha1-antagonists currently prescribed in the treatment of BPH do not exhibit in vitro selectivity between alpha1a-, alpha1b- and alpha1d-subtypes and yet they have good clinical tolerance in terms of low incidence of cardiovascular effects. One possibility to account for these findings is that another alpha1-adrenoceptor subtype could be implicated in human prostatic smooth muscle contraction. A recent report, although confirming the presence of an alpha1a-subtype in human prostate, suggested that an alpha1-adrenoceptor subtype with lower affinity for prazosin, designated alpha1L, which has not been cloned yet, is in fact the predominant alpha1-subtype involved in the contractile response of human prostatic smooth muscle to noradrenaline.

25 years since the discovery of presynaptic receptors: present knowledge and future perspectives.

The release of neurotransmitters is modulated through presynaptic autoreceptors that are acted upon by the neurone's own transmitter. The presynaptic inhibitory terminal autoreceptors were first described for noradrenergic neurones (alpha 2-adrenoceptors) and subsequently for other neurotransmitters: dopamine (D2/D3), acetylcholine (M2), GABA (GABAB), histamine (H3) and serotonin (5-HT10). This negative feedback loop introduces terminal regulation into the neural secretory event. Saloman Langer describes how presynaptic terminal facilitatory receptors exist for the modulation of acetylcholine release and these autoreceptors are of the nicotinic subtype. Presynaptic release-modulating receptors represent targets for pharmacological intervention by exogenous compounds acting as agonists, partial agonists or antagonists. Compounds with such properties as the antidepressant mirtazepine (alpha 2-adrenoceptor antagonist) and the neuroleptic amisulpride (preferential presynaptic D2/D3 receptor antagonist) may represent the beginning of a new generation of innovative drugs with useful therapeutic properties.

Effects of zolpidem on local cerebral glucose metabolism during non-REM sleep in normal volunteers: a positron emission tomography study.

Using positron emission tomography with fluorodeoxyglucose (18FDG or FDG), we compared the effects of zolpidem (10 mg), an imidazopyridine hypnotic, which is relatively selective for the BZ1 or omega receptor and placebo on cerebral glucose metabolism during the first non-REM sleep period of 12 young normal volunteers. Plasma zolpidem pharmacokinetics varied considerably among subjects, and plasma concentrations were lower than usually reported. In general, the effects of zolpidem on local cerebral glucose metabolism varied directly with plasma concentrations of zolpidem. Zolpidem induced changes in local cerebral glucose metabolism were unevenly distributed throughout the brain and were greater in subcortical areas than lateral cortical areas. Significant negative correlations were found between change in local absolute glucose metabolic rate (calculated by subtracting individual data on placebo nights from that on zolpidem nights) and plasma concentration of zolpidem for the following areas: medial frontal cortex, cingulate gyrus, putamen, thalamus, and hippocampus. The effects of zolpidem on local cerebral glucose metabolism were partially but not closely related to the reported density of BZ1 receptors.

Pharmacological characterization of the cloned human 5-hydroxytryptamine transporter.

We performed an extensive pharmacological study of the 5-hydroxytryptamine (5-HT) transporter polypeptide cloned from human placenta. Transient expression of this 630 amino acid polypeptide in HeLa cells led to saturable 5-HT uptake activity (Km = 858 nM). This 5-HT uptake was blocked by selective 5-HT inhibitors, such as citalopram, litoxetine, sertraline, and indalpine, with Ki values in the low nanomolar range, and it exhibited a pharmacological profile similar to that found in rat brain. [3H]Citalopram binding to membrane preparations of the transfected cells occurred to a single class of high-affinity binding sites (Kd = 5.3 nM) and was potently inhibited by selective 5-HT uptake inhibitors. The pharmacological profile of [3H]citalopram binding to these transfected cells showed a good correlation with that of [3H]paroxetine binding to the rat cerebral cortical 5-HT transporter (r = 0.79). These data confirm that the full pharmacological characteristics of the 5-HT transport system are conferred by the expression of the 630 amino acid human placental 5-HT transporter polypeptide. [3H]Citalopram should, therefore, provide a useful probe for more insights at a molecular level into this cloned 5-HT transport system.

Pharmacological profile of benzodiazepine site ligands with recombinant GABAA receptor subtypes.

Using [3H]flumazenil as a probe we investigated how benzodiazepine site pharmacology of alpha beta gamma ternary combinations of GABAA receptors can be influenced upon expression of different isoforms of alpha, beta and gamma subunits. The nature of the beta subunit did not alter the pharmacology of this site in that the affinities of alpha 5-containing GABAA receptors for various benzodiazepine modulatory ligands were essentially unchanged upon a comparison of different beta-variant forms (alpha 5 beta 1 gamma 2, alpha 5 beta 2 gamma 2 and alpha 5 beta 3 gamma 2). In contrast, both alpha and gamma variants contributed to notable differences in benzodiazepine site pharmacology. Thus alpha 1 beta 2 gamma 2, alpha 3 beta 2 gamma 2 and alpha 5 beta 2 gamma 2 receptors showed high, intermediate and low affinities for zolpidem, respectively. Exchanging gamma 2 for gamma 3 reduced the affinities of alpha 1 beta 2 gamma and alpha 3 beta 2 gamma receptors for zolpidem by factors of > 150 and > 5.8, respectively. The alpha 1 beta 2 gamma 3, alpha 3 beta 2 gamma 3 and alpha 5 beta 2 gamma 3 receptors exhibited, in contrast, higher affinity for CL218872 than their corresponding gamma 2 receptors. The information on these different recombinant GABAA receptor pharmacological profiles should help in the elucidation of native GABAA receptor subtype diversity.

Normalization of insulin secretion by a selective alpha 2-adrenoceptor antagonist restores GLUT-4 glucose transporter expression in adipose tissue of type II diabetic rats.

Noninsulin-dependent mellitus is characterized by the coexistence of defective insulin secretion with insulin resistance in peripheral tissues. Therapeutic objectives are, therefore, to normalize glucose-induced insulin secretion and to restore normal glucose transport into insulin-sensitive tissues. In the present study we evaluate the effects of acute and subchronic administration (2 or 10 days) of the alpha 2-adrenoceptor antagonist SL 84.0418 on glucose tolerance in nondiabetic control rats and type I and type II diabetic rats and the level of the insulin-sensitive glucose transporter GLUT-4, which is exclusively expressed in white and brown adipose tissues, heart, and skeletal muscles. Glucose tolerance and insulin secretion were markedly impaired in type II diabetic rats (neonatal injection of streptozotocin) and were totally corrected by an acute i.p. injection of SL 84.0418. As a consequence of the chronic restoration of insulin secretion, GLUT-4 messenger RNA (mRNA) levels, initially decreased by 67% in white adipose tissue of type II diabetic rats, were normalized by subchronic (10 days), but not acute (2 days), treatment with SL 84.0418. The same results were obtained in brown adipose tissues of type II diabetic rats, whereas no modification of GLUT-4 mRNA levels remained very low in brown adipose tissues of type I diabetic rats (adult injection of streptozotocin) after acute or subchronic administration of SL 84.0418, suggesting that this drug acted by the restoration of insulin secretion. This study reports a decrease in GLUT-4 levels in insulin-sensitive tissues in this model of type II diabetes as well as its regulation after subchronic normalization of insulin secretion. We suggest a direct role for the alpha 2-adrenoceptor antagonist SL 84.0418 in pancreatic beta-cells that allows normalization of glucose tolerance.

Antagonist properties of the stereoisomers of ifenprodil at NR1A/NR2A and NR1A/NR2B subtypes of the NMDA receptor expressed in Xenopus oocytes.

The NMDA receptor antagonist ifenprodil contains two asymmetric centres which give rise to four stereoisomeric forms of this molecule. The inhibitory effects of each of these stereoisomers on recombinant NMDA receptors expressed from NR1A/NR2A and NR1A/NR2B subunit combinations were studied in Xenopus oocytes by voltage-clamp recording. All four ifenprodil stereoisomers were potent antagonists at NR1A/NR2B (IC50 < 0.8 microM), but weak antagonists at NR1A/NR2A receptors (IC50 > 100 microM). In heteromeric NR1A/NR2B receptors, (+) erythro- and (-) threo-ifenprodil (IC50 0.21 and 0.22 microM, respectively) were about 4 times more potent than (-) erythro- and (+) threo-ifenprodil (IC50 0.81 and 0.76, respectively). These results show that the stereoisomers of ifenprodil exhibit a weak though significant stereoselectivity at the NR1A/NR2B NMDA receptor subtype.

Quantification of alpha 1-adrenoceptor subtypes in human tissues by competitive RT-PCR analysis.

Alpha 1-Adrenoceptors are a heterogeneous subfamily of receptors comprising at least three pharmacologically- and structurally- distinct subtypes named alpha 1a-, alpha 1b- and alpha 1d-adrenoceptors. A competitive reverse transcription-polymerase chain reaction methodology using subtype selective competitor RNAs as reference internal standards was adopted to determine the absolute amounts of each of the three cloned to date alpha 1-adrenoceptor subtype encoding mRNAs in selected human tissues. Our data demonstrated that each of these alpha 1-adrenoceptor subtypes exhibits a distinctive tissular expression pattern.

Modulation of the gamma-aminobutyric acid type A receptor by the antiepileptic drugs carbamazepine and phenytoin.

We report here that carbamazepine and phenytoin, two widely used antiepileptic drugs, potentiate gamma-aminobutyric acid (GABA)-induced Cl- currents in human embryonic kidney cells transiently expressing the alpha 1 beta 2 gamma 2 subtype of the GABAA receptor and in cultured rat cortical neurons. In cortical neuron recordings, the current induced by 1 microM GABA was enhanced by carbamazepine and phenytoin with EC50 values of 24.5 nM and 19.6 nM and maximal potentiations of 45.6% and 90%, respectively. The potentiation by these compounds was dependent upon the concentration of GABA, suggesting an allosteric modulation of the receptor, but was not antagonized by the benzodiazepine (omega) modulatory site antagonist flumazenil. Carbamazepine and phenytoin did not modify GABA-induced currents in human embryonic kidney cells transiently expressing binary alpha 1 beta 2 recombinant GABAA receptors. The alpha 1 beta 2 recombinant is known to possess functional barbiturate, steroid, and picrotoxin sites, indicating that these sites are not involved in the modulatory effects of carbamazepine and phenytoin. When tested in cells containing recombinant alpha 1 beta 2 gamma 2, alpha 3 beta 2 gamma 2, or alpha 5 beta 2 gamma 2 GABAA receptors, carbamazepine and phenytoin potentiated the GABA-induced current only in those cells expressing the alpha 1 beta 2 gamma 2 receptor subtype. This indicates that the nature of the alpha subunit isoform plays a critical role in determining the carbamazepine/phenytoin pharmacophore. Our results therefore illustrate the existence of one or more new allosteric regulatory sites for carbamazepine and phenytoin on the GABAA receptor. These sites could be implicated in the known anticonvulsant properties of these drugs and thus may offer new targets in the search for novel antiepileptic drugs.

Further evidence that the classical alpha 1A- and cloned alpha 1c-adrenoceptors are the same subtype.

We compared the inactivation of [3H]prazosin binding sites in membrane preparations of cell-lines expressing the cloned alpha 1b, alpha 1c and alpha 1d-adrenoceptors after pretreatment with the alkylating agents, 10 microM chlorethylclonidine or 10 nM SZL-49 (1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-bicyclo[2.2.2]octa- 2,5-diene-Z-carbonyl)-piperazine). The cloned alpha 1b-adrenoceptor exhibited a similar inactivation profile to that of the classical alpha 1B-adrenoceptor of rat liver in that chlorethylclonidine in contrast to SZL-49 produced a marked degree of inactivation. A similarity between the cloned alpha 1c-adrenoceptor and the classical alpha 1A-adrenoceptor of rat submaxillary gland was also noted in that both subtypes were highly sensitive to SZL-49 but relatively insensitive to chlorethylclonidine. The cloned alpha 1d-adrenoceptor displayed a unique profile in that both chlorethylclonidine and SZL-49 produced a marked inactivation of this subtype. The similarity of the alkylation-inactivation profiles between the cloned alpha 1c and classical alpha 1A-adrenoceptors support the recent proposal that these two alpha 1-adrenoceptors in fact correspond to the same subtype.