Modulation of agonist stimulated adenylyl cyclase and GTPase activity by L-pro-L-leu-glycinamide and its peptidomimetic analogue in rat striatal membranes.

L-prolyl-L-leucyl-glycinamide (PLG), also known as melanocyte-stimulating hormone release inhibiting factor (MIF-1), is an endogenous brain tripeptide. Previous studies have shown that PLG, and its peptidomimetic analogues, render dopamine D2 receptors more responsive to agonists by maintaining the high-affinity binding state of the receptors. In the present study, we examined the effect PLG and its analogue 3(R)-[(2(S)-pyrrolidylcarbonyl)amino]-2-oxo-1-pyrrolidineacetam ide (PAOPA) on dopamine-stimulated adenylyl cyclase and NPA-stimulated GTPase activity in rat striatal membranes. Dopamine-stimulated adenylyl cyclase activity was inhibited by both PLG and PAOPA in a dose-dependent manner, whereas R(-)-propylnorapomorphine (NPA)-stimulated low Km GTPase activity was significantly increased by 1 microM PLG or 1 nM PAOPA. These results suggest that PLG and PAOPA maintain the high affinity state of the D2 receptor by increasing GTP hydrolysis through stimulation of agonist-induced GTPase activity.

Properties of mu 3 opiate alkaloid receptors in macrophages, astrocytes, and HL-60 human promyelocytic leukemia cells.

An opiate alkaloid-selective receptor, designated mu(3), mediates inhibition by morphine of activation of human peripheral blood monocytes and granulocytes. The mu(3) receptor is present on several macrophage cell types including microglia, on cultured astrocytes, and in brain and retina. Murine macrophage cell lines and human HL-60 leukemia cells contain high concentrations of these receptors. Binding of 3H-morphine to the receptor is displaced by morphine, etorphine, naloxone, diprenorphine and morphine 6-glucuronide, but not by morphine 3-glucuronide, fentanyl, benzomorphans, enkephalins, dynorphin, beta-endorphin, endomorphin-1, other opioid peptides or nociceptin (orphanin FQ). The mu(3) receptor appears to be much more sensitive to inactivation by reduced glutathione than are classical mu, delta and kappa receptors. Evidence is also presented for G protein-coupling of these receptors. These and other data raise the possibility that the mu(3) receptor is a member of a chemokine or of another related receptor family, rather than the opioid receptor family. The affinity for morphine of mu(3) receptors of granulocytic-differentiated HL-60 cells is markedly enhanced in the presence of levorphanol and certain benzomorphans. In contrast, receptors of monocytes, macrophage cell lines, microglia, macrophage-differentiated HL-60 cells and astrocytes are not affected by levorphanol or benzomorphans. It is concluded that mu(3) receptors of granulocytic and promyelocytic cells differ from those of macrophage and astrocyte cell types, possibly due to differences in receptor subtype or to the presence of an additional component in the granulocytic and promyelocytic cells.

Presence and characterization of nociceptin (orphanin FQ) receptor binding in adult rat and human fetal hypothalamus.

High affinity and saturable nociceptin (orphanin FQ) receptors were detected and characterized in adult rat and human fetal hypothalamic membranes, utilizing [125I]Tyr12-nociceptin as ligand. Nociceptin bound with picomolar affinity, dynorphin A with nanomolar affinity, naloxone and dynorphan A(1-8) with micromolar while des-Tyr1-dynorphin (dynorphin A(2-17)), several other opioids, morphine and benzomorphans failed to compete for binding at 1-10 microM. Gpp(NH)p together with sodium ion markedly decreased binding, consistent with involvement of a G protein-linked receptor.

Presence of nociceptin (orphanin FQ) receptors in rat retina: comparison with receptors in striatum.

Nociceptin (orphanin FQ), a heptadecapeptide with some sequence homology to dynorphin A, has been proposed as an endogenous ligand for a previously cloned orphan receptor with significant homology to opioid receptors. Utilizing [(125)I][Tyr14]nociceptin as ligand, saturable and high affinity nociceptin binding sites were detected and characterized in rat retina and striatum. For retina, Bmax = 44.0 +/- 4.5 fmol/mg and Kd = 32.4 +/- 2.7 pM; for striatum, Bmax = 51.6 +/- 7.7 fmol/mg and Kd = 98.6 +/- 11.3 pM. In competition studies, nociceptin bound with picomolar affinity, dynorphin A with nanomolar affinity, naloxone and dynorphan A-(1-8) with micromolar affinity, while [des-Tyr1]dynorphin (dynorphin A-(2-17)), several other opioids, morphine and benzomorphans failed to compete for binding at 1-10 microM. Gpp(NH)p plus NaCl markedly decreased binding, consistent with involvement of a G protein-linked receptor. It is concluded that rat retina contains nociceptin receptors similar in concentration to those present in striatum. Properties of both the retinal and the striatal receptors are similar to those previously found for rat hypothalamus.

Opioid and opiate immunoregulatory processes.

The discovery of the ability of the nervous system to communicate through "public" circuits with other systems of the body is attributed to Ernst and Berta Scharrer, who described the neurosecretory process in 1928. Indeed, the immune system has been identified as another important neuroendocrine target tissue. Opioid peptides are involved in this communication (i.e., neuroimmune) and with that of autoimmunoregulation (communication between immunocytes). The significance of opioid neuropeptide involvement with the immune system is ascertained from the presence of novel delta, mu, and kappa receptors on inflammatory cells that result in modulation of cellular activity after activation, as well as the presence of specific enzymatic degradation and regulation processes. In contrast to the relatively uniform antinociceptive action of opiate and opioid signal molecules in neural tissues, the presence of naturally occurring morphine in plasma and a novel mu3, opiate-specific receptor on inflammatory cells adds to the growing knowledge that opioid and opiate signal molecules may have antagonistic actions in select tissues. In examining various disorders (e.g., human immunodeficiency virus, substance abuse, parasitism, and the diffuse inflammatory response associated with surgery) evidence has also been found for the involvement of opiate/opioid signaling in prominent mechanisms. In addition, the presence of similar mechanisms in man and organisms 500 million years divergent in evolution bespeaks the importance of this family of signal molecules. The present review provides an overview of recent advances in the field of opiate and opioid immunoregulatory processes and speculates as to their significance in diverse biological systems.

Occurrence of the opiate alkaloid-selective mu3 receptor in mammalian microglia, astrocytes and Kupffer cells.

Evidence is presented for occurrence of opiate alkaloid-selective, opioid-peptide-insensitive receptor binding sites, labeled with [3H]morphine, in primary cultures of cat microglia and cat astrocytes, as well as on highly purified preparations of rat Kupffer cells. These receptors have been designated mu3 on the basis of their close similarity to receptors first found to be present on human peripheral blood monocytes. Exposure of the microglia to morphine and etorphine caused marked quantifiable changes in cellular morphology, including assumption of a more rounded shape and retraction of cytoplasmic processes; in contrast, several opioid peptides were without effect on morphology. The effects of morphine on microglial morphology were blocked by the opiate antagonist naloxone. These effects of drugs on morphology were as predicted for action via the mu3 receptor. Opiate alkaloid binding sites previously detected on the rat C6 glioma cell line were also characterized here as of the mu3 receptor subtype. It is proposed that mu3 receptors have broad distribution in different macrophage cell types of bone marrow lineage, including microglia and Kupffer cells. Furthermore, these receptors are not restricted to cells of bone marrow lineage, since they are also present on astrocytes.

Human granulocytes contain an opiate alkaloid-selective receptor mediating inhibition of cytokine-induced activation and chemotaxis.

Human peripheral blood granulocytes previously were found to contain opioid delta 2-receptors mediating stimulation by opioid peptides of chemotaxis. Studies presented in this work indicate that granulocytes also contain opiate alkaloid-selective, opioid peptide-insensitive receptors mediating inhibition by morphine and other opiates of cytokine-induced activation and chemotaxis. Binding studies with [3H]morphine and [3H]diprenorphine ([3H]DPN) indicated the presence of receptor sites, at considerable density with affinities and selectivity for opiates comparable with those of the mu 3-receptor of human peripheral blood monocytes (macrophages). The influence of the guanosine 5'-triphosphate (GTP) analogue GppNHp on binding indicated that the granulocyte receptor was linked to a G protein. Morphine but not opioid peptides interfered with activation and/or chemotaxis of the granulocytes induced by TNF-alpha, IL-1 alpha, IL-8, and FMLP (chemotactic peptide). These effects of morphine were blocked by the antagonist naloxone. Levorphanol inhibited TNF-alpha-induced activation, and also potentiated the inhibition by morphine. Furthermore, in binding assays, levorphanol enhanced the affinity of the receptor for morphine. Dextrorphan had no effect on activation or chemotaxis, and it also had no effect on binding, indicative of stereoselectivity for the effect of levorphanol. It is concluded that human granulocytes contain opiate alkaloid-selective mu 3-receptors that mediate inhibitory effects of morphine on cellular activation by cytokines.

Murine macrophage cell lines contain mu 3-opiate receptors.

Opiate alkaloid-selective, opioid peptide-insensitive mu 3 receptors are present in three murine macrophage cell lines (J774.2; RAW 264.7; BAC1.2F5). The receptor binds morphine, its active metabolite morphine 6-glucuronide and certain other alkaloids, but not morphine 3-glucuronide or any of the opioid peptides tested. The cell lines thus provide valuable model systems for investigation of mu 3-opiate receptors, previously demonstrated to mediate inhibitory effects of morphine on activation of human peripheral blood macrophages (monocytes).

GABAA receptor activation induces GABA and glutamate release from preoptic area.

The effect of GABA receptor agonists on release in vitro of radiolabeled GABA and glutamate was studied using a crude preparation of isolated nerve terminals (neurosomes). GABA agonists were incubated (2 min, 37 degrees C) with neurosomes prepared from hypothalamus, preoptic area (POA) and frontal cortex tissues. Under these conditions, GABA and the GABAA receptor agonist muscimol, but not the GABAB receptor agonist baclofen, stimulated 3H-GABA and 3H-glutamate release from POA but not hypothalamic or cortical neurosomes of gonadally intact male rats. These effects were inhibited by the GABAA receptor antagonists picrotoxin, bicuculline and SR-95531. Significant efflux of 3H-glutamate could be elicited from cortical neurosomes following longer (5 min) incubations with 500 microM GABA and 400 microM muscimol. Muscimol-induced release of 3H-glutamate and 3H-GABA was dependent on extracellular calcium. Muscimol and GABA failed to release 3H-GABA or 3H-glutamate from POA neurosomes of ovariectomized female rats. However, administration of estradiol and progesterone to ovariectomized females prior to sacrifice caused the appearance of muscimol induced-release of amino acids from POA neurosomes comparable to that obtained in male rats. GABA-induced release of 3H-glutamate was similarly dependent on pretreatment of ovariectomized rats with ovarian steroids. GABAA receptor-induced release of amino acids is therefore brain region-specific and modified by hormonal status.

Presence in neuroblastoma cells of a mu 3 receptor with selectivity for opiate alkaloids but without affinity for opioid peptides.

Evidence is presented for the occurrence of a unique opiate alkaloid-selective, opioid peptide-insensitive binding site in N18TG2 mouse neuroblastoma cells and in late passage hybrid F-11 cells, derived from N18TG2 neuroblastoma cells and rat dorsal root ganglion cells. Those cells lacked classical opioid peptide-sensitive receptor subtypes, but contained [3H]morphine and [3H]diprenorphine binding sites with affinity for certain opiate alkaloids but not for any endogenously occurring opioid peptide or peptide analog tested, including D-ala2-D-leu5-enkephalin (DADLE), D-Ala2,N-Me-Phe4,Gly5-ol (DAGO) and dynorphin A(1-17). The binding site differed from hitherto described mu, delta and kappa neuronal opioid receptors not only on the basis of peptide insensitivity, but also on the basis of selectivity and affinities of alkaloids. Saturation experiments with [3H]morphine indicated the presence of a single site with Kd = 49 nM and Bmax = 1510 fmol/mg protein. This novel binding site was not present in F-11 hybrid cells at early passage. Instead the hybrid cells contained conventional opioid receptors (predominantly delta and also mu) capable of binding DADLE and other peptides as well as opiate alkaloids. With additional passage (cell divisions) of the hybrid cells, during which a limited change occurred in mouse chromosome number, the peptide-insensitive binding appeared and the opioid peptide-binding (delta and mu) receptors were lost reciprocally. Thus, expression of the peptide-insensitive binding normally may be repressed when conventional opioid receptors are expressed. The peptide-insensitive opiate binding site described here appears to correspond to the mu 3 receptor subtype, recently identified pharmacologically and functionally in several cell types of the immune system.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphine receptors in immunocytes and neurons.

Receptor interactions of morphine are reviewed, with particular attention given to a recently discovered opiate receptor, designated mu 3, with unique selectivity for morphine and certain other opiate alkaloids. Morphine, other opiate alkaloids and related analogs are known to bind to the classical delta, mu and kappa opioid receptor subtypes. Each of these subtypes also binds one or more of the endogenous opioid peptides with high affinity. Immunocytes have recently been found to contain a unique receptor for morphine, capable of binding morphine and certain other opiate alkaloids, but with essentially no or exceedingly low affinity for the naturally occurring endogenous opioid peptides or peptide analogs. This putative mu 3 (morphine/opiate alkaloid) receptor is present in invertebrate immunocytes as well as in human peripheral blood monocytes (macrophages). More recently this same receptor has been found in certain established macrophage cell lines and in human peripheral blood granulocytes. Finally, the same or closely related opiate alkaloid-selective (mu 3) receptor has been found to be present in a neuroblastoma and in a hybrid neural cell line. Studies indicate that in the immunocytes the receptor mediates inhibitory effects of morphine on cellular chemotaxis. While the functional coupling of this receptor in neurons is not known, it is postulated that the receptor may mediate effects of opiates on neuronal differentiation and cell division as well as neuronal transmission. Both for the immune system and the nervous system, the mu 3 receptor may constitute a major site of action for putative endogenous morphine or morphine-like substances. This receptor system also provides an additional pharmacological site of action for exogenously administered opiate alkaloid drugs. The mu 3 receptor is proposed to be an important neuro-immune link. This system is likely to play a significant role in a variety of responses involving the immune system, including the response of the organism to stress, infection and malignant transformation.

Estrogen uncouples beta-adrenergic receptor from the stimulatory guanine nucleotide-binding protein in female rat hypothalamus.

The responsiveness of adenylyl cyclase to beta-adrenergic receptor stimulation was investigated in membranes prepared from hypothalamus-preoptic area and cortex of ovariectomized female rats injected with oil vehicle or estradiol benzoate 24 or 48 h before death. Membranes from the hypothalamus-preoptic area of ovariectomized animals displayed a concentration-dependent stimulation of adenylyl cyclase when incubated with the beta-adrenergic receptor agonist, isoproterenol (10(-7)-10(-5) M). This response was suppressed in membranes from estrogen-treated animals. The effect of estrogen was observed 48 h, but not 24 h, after hormone administration. In addition, estrogen had no measurable effect on hypothalamic adenylyl cyclase activation by either GTP (10(-8)-10(-5) M) or forskolin (10(-8)-10(-6) M), on beta-adrenergic receptor density, or on antagonist binding affinity measured with the beta-adrenergic antagonist [125I]iodocyanopindolol. Analysis of isoproterenol displacement of iodocyanopindolol binding revealed that estrogen reduced agonist binding affinity in hypothalamus-preoptic area membranes. In membranes from ovariectomized controls, high affinity agonist binding to the beta-adrenergic receptor was apparent and was abolished by guanine nucleotides. However, membranes from estradiol-treated rats demonstrated only low affinity agonist binding that was unaffected by guanine nucleotides. Estradiol did not detectably alter concentrations of either cholera or pertussis toxin substrates in hypothalamus-preoptic area membranes. These data indicate that estrogen promotes a stable time-dependent desensitization of beta-adrenergic receptor activation of adenylyl cyclase in hypothalamus and preoptic area by uncoupling the receptor from the guanine nucleotide-binding protein, G8.

Opiate-like substances in an invertebrate, an opiate receptor on invertebrate and human immunocytes, and a role in immunosuppression.

The presence of morphine-like and codeine-like substances was demonstrated in the pedal ganglia, hemolymph, and mantle tissues of the mollusc Mytilus edulis. The pharmacological activities of the endogenous morphine-like material resemble those of authentic morphine. Both substances were found to counteract, in a dose-dependent manner, the stimulatory effect of tumor necrosis factor alpha or interleukin 1 alpha on human monocytes and Mytilus immunocytes, when added simultaneously to the incubation medium. The immunosuppressive effect of this opiate material expresses itself in a lowering of chemotactic activity, cellular velocity, and adherence. Codeine mimics the activity of authentic morphine, but only at much higher concentrations. Specific high-affinity receptor sites (mu 3) for morphine have been identified on human monocytes and Mytilus immunocytes. In Mytilus recovering from experimentally induced stress, the return of "altered" immunocytes to a more inactive state appears to be due to a significant rise in the content of morphine-like material in the pedal ganglia and hemolymph at this time. Thus, morphine may have a role in calming or terminating the state of immune alertness.

Direct coupling of opioid receptors to both stimulatory and inhibitory guanine nucleotide-binding proteins in F-11 neuroblastoma-sensory neuron hybrid cells.

Evidence is presented for linkage of opioid receptors directly to the stimulatory G protein (guanine nucleotide-binding protein), Gs, in addition to the generally accepted linkage to the inhibitory and "other" G proteins, gi and Go, in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Treatment of intact F-11 cells with cholera toxin decreased specific binding of the opioid agonist [D-Ala2,D-Leu5]enkephalin to F-11 cell membranes by 35%, with the remaining binding retaining high affinity for agonist. Under these conditions cholera toxin influenced the alpha subunit of Gs (Gs alpha) but had no effect on the alpha subunit of Gi/o (Gi/o alpha), based on ADP-ribosylation studies. Pertussis toxin treatment decreased high-affinity opioid agonist binding by about 50%; remaining binding was also of high affinity, even though pertussis toxin had inactivated Gi/o alpha selectively and essentially completely. Simultaneous treatment with both toxins had an additive effect, reducing specific binding by about 80%. While opioid agonists inhibited forskolin-stimulated adenylate cyclase activity of F-11 cells as expected, opioids also stimulated basal adenylate cyclase activity, indicative of interaction with Gs as well as Gi. Cholera toxin treatment attenuated opioid-stimulation of basal adenylate cyclase, whereas pertussis toxin treatment enhanced stimulation. In contrast, inhibition by opioid of forskolin-stimulated activity was attenuated by pertussis toxin but not by cholera toxin. It is concluded that a subset of opioid receptors may be linked directly to Gs and thereby mediate stimulation of adenylate cyclase. This Gs-adenylate cyclase interaction is postulated to be responsible for the novel excitatory electrophysiologic responses to opioids found in our previous studies of sensory neurons and F-11 cells.

Characterization of glutamate efflux from preoptic area synaptosomes.

Treatment of ovariectomized rats in vivo with ovarian steroids has been found to influence the efflux of glutamate and gamma-aminobutyric acid from preoptic area synaptosomes incubated in vitro. Since these studies indicated a possible role of the glutamate carrier in steroid-modulated release of amino acids, the present studies examined the characteristics of efflux of glutamate and of the carrier system for glutamate in synaptosomes of the preoptic area derived from ovariectomized hormone-treated rats. The efflux of [3H]glutamate from preoptic area synaptosomes, was induced by glutamate and by the glutamate carrier agonist, D-aspartate; the putative glutamate carrier antagonist dihydrokainate failed to block this efflux. Dihydrokainate inhibited the uptake of glutamate but it was less effective than D-aspartate. The excitatory amino acid receptor agonists, N-methyl-D-aspartate and kainate were without effect while quisqualate modestly stimulated the efflux of [3H]glutamate. Efflux of [3H]glutamate, induced by glutamate itself or by D-aspartate was not blocked by the excitatory amino acid receptor antagonists, D-2-amino-5-phosphonovaleric acid, 6,7-dinitroquinoxaline-2,3-dione or kynurenate. Glutamate-induced efflux of [3H]glutamate did not require external Ca2+. Glutamate altered neither the basal nor the potassium-induced increases in the intrasynaptosomal concentration of Ca2+ as measured by the fura-2 method. Glutamate-induced efflux of [3H]glutamate was blocked by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. It is concluded that the glutamate-induced efflux of [3H]glutamate in synaptosomes of the preoptic area is a carrier-mediated process that does not require activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Estradiol plus progesterone promote glutamate-induced release of gamma-aminobutyric acid from preoptic area synaptosomes.

Treatment of ovariectomized rats with both estradiol and progesterone in vivo resulted in a marked enhancement of glutamate-induced release of newly synthesized [3H]gamma-aminobutyric acid (GABA) from synaptosomes of the preoptic area in vitro. With this treatment, as little as 0.01 nM glutamate, in vitro, enhanced release of GABA. In contrast, glutamate, in vitro, did not stimulate release of GABA from synaptosomes, obtained from rats treated with either estradiol or progesterone alone and only large concentrations of glutamate (1.0 and 10 mM) caused a modest release of GABA from synaptosomes from ovariectomized, vehicle-treated rats. Also, treatment with estradiol plus progesterone did not alter glutamate-induced release or exchange of [3H]glutamate. Glutamate-induced release of GABA was calcium-independent and attenuated by the putative chloride channel antagonist, 4,4'-diisothiocyanatostilbene-2,2'-DL-disulfonic acid. Thus, glutamate-induced, steroid-enhanced release of GABA may occur through a chloride-dependent carrier rather than by exocytosis. In addition to enhancement by glutamate, release of GABA was also enhanced by D-aspartate, an agent that is transported by the neuronal glutamate carrier. It is postulated that enhancement of glutamate-induced release of GABA, by estradiol plus progesterone in the preoptic area, represents one process by which these steroids modulate reproductive function in female rats.

2-amino-4-phosphonobutyric acid exerts a light-dependent effect on post-gabaculine levels of retinal gamma-aminobutyric acid (GABA): evidence that ON synaptic pathways regulate retinal GABAergic transmission.

The effects of light, 2-amino-4-phosphonobutyric acid (APB), and kainic acid on rat retinal gamma-aminobutyric acid (GABA)-ergic transmission were studied by measuring levels of retinal GABA following subcutaneous injection of gabaculine, an irreversible inhibitor of GABA-transaminase. Post-gabaculine levels of retinal GABA in light-exposed rats were significantly greater than those in rats held in darkness. The synaptic mechanism of this effect of light was examined by measuring post-gabaculine levels of retinal GABA in rats placed into either lighted or darkened conditions after receiving unilateral intravitreal injections of APB, a glutamate analogue that selectively decreases the activity of ON synaptic pathways in the retina. APB attenuated the post-gabaculine accumulation of GABA in rats held in the light, but not in those placed into darkness. Furthermore, the light-dependent increment in post-gabaculine accumulation of retinal GABA was entirely APB sensitive, and the effect of APB was entirely light dependent. In contrast to APB, kainic acid stimulated the post-gabaculine accumulation of retinal GABA in vivo. Our findings suggest that APB and kainic acid influence GABAergic transmission at different sites in the retina and that some retinal GABAergic neurons are either ON or ON-OFF amacrine cells.

Ovarian steroids increase veratridine-induced release of amino acid neurotransmitters in preoptic area synaptosomes.

In vivo treatment of ovariectomized rats with estradiol benzoate plus progesterone, but not with either steroid alone, produced a large increase in veratridine-induced release of radiolabeled glutamate and newly synthesized GABA from preoptic area synaptosomes in vitro. Neither basal nor KCl-evoked release of amino acids was altered. Thus gonadal steroids appear to be involved in the control of amino acid neurotransmitter release in a brain region of importance for regulation of female reproductive physiology and behavior.

The effects of gabaculine in vivo on the distribution of GABA-like immunoreactivity in the rat retina.

gamma-Aminobutyric acid (GABA) is an inhibitory transmitter found in the retinae of mammals largely within certain amacrine cells. In previous studies from this laboratory, subcutaneous administration to rats of gabaculine, an enzyme-activated irreversible inhibitor of gamma-aminobutyric acid (GABA)-transaminase, produced large, rapid and long-lasting increases in levels of retinal GABA. We employed immunocytochemistry to determine whether such changes in the levels of retinal GABA are accompanied by changes in the cellular distribution of GABA. Using a recently developed antiserum to a GABA-protein conjugate, and the peroxidase-antiperoxidase method, we examined retinae from control rats and from rats 2 or 8 h after administration of 10 mg/kg gabaculine. From previous work, retinal levels of GABA were respectively elevated 3- or 6-fold at those postgabaculine times. In the present study, marked changes in the distribution of GABA-like immunoreactivity (GABA-LIR) were apparent by 2 h after injection of gabaculine, and were more striking at 8 h postgabaculine. The pattern of staining for GABA-LIR strongly suggested that much of the GABA in gabaculine-treated retinae was within Müller glial cells. That observation provides evidence for the importance of those cells in the uptake and degradation of GABA after its release from retinal neurons.