Basal cell adenocarcinoma in the gland of the third eyelid of a brown bear (Ursus arctos).

The right third eyelid of an adult female brown bear (Ursus arctos) was swollen and removed. Histopathology revealed a tumor exhibiting proliferation with mild infiltration, consisting of multi-stratified glandular structures of the innermost laminal neoplastic cells and the basaloid neoplastic cells, and with eosinophilic thick basal lamina material around the glandular structures. Both types of neoplastic cells exhibited moderate anisokaryosis, and mitotic figures were observed in the basaloid neoplastic cells. The laminal neoplastic cells were cytokeratin (CK) 8/18-positive. In contrast, the basaloid neoplastic cells were CK14- and p63-positive, but α-smooth muscle actin- and calponin-negative. The case described herein is the first report of basal cell adenocarcinoma in the gland of the third eyelid of a bear.

Histopathological and immunohistochemical features of atypical epithelial tumours of the gland of the third eyelid in seven dogs.

This report documents the histopathological and immunohistochemical features of atypical epithelial tumours of the gland of the third eyelid (GTE) in seven dogs. Cases 1 and 2 were diagnosed as myoepithelioma, comprising of compressive proliferations of interlacing bundles of neoplastic spindle cells expressing cytokeratin 14, p63, calponin and α-smooth muscle actin. Cases 3, 4 and 5 were diagnosed as complex carcinomas comprising of atypical glandular cells expressing cytokeratin 8/18, together with spindle-shaped or round neoplastic cells expressing cytokeratin 14, p63, calponin and α-smooth muscle actin. Cases 6 and 7 were diagnosed as basal cell adenocarcinomas (BCACs) comprising of a mixed proliferation of glandular and basal-type cells expressing cytokeratin 14 and p63. Therefore, in addition to glandular components, these tumours may include neoplastic cells with a myoepithelial or basal cell phenotype. Hence, there is diversity in the features of epithelial neoplasia of the GTE in dogs, similar to tumours in human salivary and lacrimal glands.

Marked depletion of the water-channel protein, AQP5, in the canine nictitating membrane glands might contribute to the development of KCS.

The objectives of this study were to investigate the normal histological localization of aquaporin (AQP) 5 protein in the lacrimal and nictitating membrane glands and to compare this localization in healthy and keratoconjunctivitis sicca (KCS) dogs. Lacrimal and nictitating membrane glands of 5 healthy Beagles and nictitating membrane glands of 5 KCS dogs (3 Beagles and 2 mongrel dogs: 0-13 years) were used for the present study. The owners of the KCS dogs did not consent to perform biopsies of the lacrimal glands. The localization and distribution of AQP5 protein were investigated by an immunohistochemical technique. In immunohistochemical staining, AQP5 was localized in the apical site of acinar epithelial and ductal epithelial cells from both the lacrimal and nictitating membrane glands in healthy dogs. However, AQP5 was not detected in the 5 KCS dogs. These results for immunohistochemical AQP5 localization might correlate with the deficiency in tear secretion found in KCS dogs.

Distribution of goblet cells and MUC5AC mRNA in the canine nictitating membrane.

This study evaluated the distribution of goblet cells and the expression of MUC5AC mRNA in the canine nictitating membrane. The distribution of goblet cells in the nictitating membrane and temporal bulbar conjunctiva of beagle dogs was examined by histochemical analysis of impression cytology specimens and frozen sections. MUC5AC mRNA was detected by the reverse transcription polymerase chain reaction (RT-PCR). The distribution of MUC5AC mRNA was also examined by in situ hybridization using digoxigenin-labeled antisense and sense RNA probes. Histochemical analysis showed that the canine nictitating membrane epithelium contained many more periodic acid-Schiff positive goblet cells, particularly on the palpebral side, compared with the temporal bulbar conjunctiva. RT-PCR revealed that MUC5AC was expressed in both the nictitating membrane and in conjunctival tissue. When the distribution of MUC5AC mRNA was assessed by in situ hybridization, its expression was high on the palpebral side of the nictitating membrane and low in the temporal bulbar conjunctiva. MUC5AC mRNA expression corresponded with the distribution of goblet cells by histochemical examination. In conclusion, there were numerous goblet cells in the canine nictitating membrane epithelium, particularly on the palpebral side, and MUC5AC mRNA was expressed in the nictitating membrane epithelium at locations corresponding to the goblet cells.

Tissue distribution of dexamethasone in canine ocular compartments following topical application of dexamethasone-21-isonicotinate and oxytetracycline HCl.

OBJECTIVE: To study the dexamethasone (DXM) concentration at different time points in various compartments of the canine eye following topical application of DXM-21-isonicotinate and oxytetracycline hydrochloride ANIMALS STUDIED: Thirty dogs to be euthanized for reasons not related to this study were selected and their ocular health status evaluated. Selected animals were treated with DXM-oxytetracycline ointment and euthanized after 6, 11 or 16 h. PROCEDURE: The concentration of DXM was determined in the following compartments of the eye: third eyelid, cornea, aqueous humor, iris, lens, vitreous body and choroid/retina. The DXM concentration in the eye was measured by radioimmunoassay. The applied amount of DXM was 0.04 mg in 0.2 mL ointment. Dogs were treated once with Corti Biciron eye ointment (DXM-21-isonicotinate and oxytetracycline hydrochloride, S & K Pharma, Perl, Germany) and were euthanized 6, 11 and 16 h after treatment. RESULTS: At 6 h following topical application the mean DXM concentration was highest in the anterior structures of the eye (third eyelid: 18 ng/g, cornea: 36 ng/g). The concentration in the posterior structures was below detection level. A decreased DXM concentration in the anterior structures was measured 11 and 16 h after treatment. CONCLUSION: It could be demonstrated that therapeutically relevant concentrations of DXM after a single topical administration are only achieved in anterior structures of the eye. A dosing interval of 6-11 h is recommended to achieve therapeutic drug concentration in those structures. The posterior structures of the eye are not reached by topical administration.

Analysis of long-term cognitive-enhancing effects of bryostatin-1 on the rabbit (Oryctolagus cuniculus) nictitating membrane response.

Previous work demonstrated that protein kinase C (PKC) is implicated in learning and memory. This study investigated whether: (i) PKC activated by bryostatin-1 (Bryo) just before or just after sessions of classical conditioning was capable of enhancing classical conditioning of the rabbit nictitating membrane response; (ii) improved behavioral performance matched the time course of PKC activation induced by Bryo; and (iii) vitamin E (Vit E) enhanced the efficacy of Bryo. Paired rabbits received daily trace conditioning with a tone conditioned stimulus and a corneal air puff unconditioned stimulus. Unpaired rabbits received the same stimuli but in an explicitly unpaired manner. After trace conditioning, all rabbits received daily delay conditioning, and then tone intensity testing. Rabbits pretreated with 10 microg/kg Bryo every other day before a relatively simple trace conditioning task showed more conditioned responses (CRs) during the first 10 trials of each trace conditioning session and a higher likelihood of a CR on the first trial of each trace conditioning session than rabbits pretreated with the vehicle control. Rabbits either posttreated daily with 10 microg/kg Bryo or pretreated with Vit E and subjected to a difficult trace conditioning task showed increased CRs relative to the vehicle control. Neither Bryo nor Vit E or their combination altered nonassociative responding or altered sensitivity to the conditioned stimulus or unconditioned stimulus. These findings demonstrate Bryo has long-term enhancing effects on classical conditioning of the rabbit nictitating membrane response.

Immunophenotyping of lymphocyte subsets in the third eyelid tissue in dogs (Canis familiaris): morphological, microvascular, and secretory aspects of this ocular adnexa.

The third eyelid is an important adnexa of the eye. The objective of this study was to evaluate (i) morphological aspects (ii) vascularization, and (iii) the immunophenotype of lymphocyte subsets in the third eyelid of dogs. Flow cytometric analysis revealed the presence of three patterns concerning the immunophenotype of the third eyelid tissue. Dogs without ocular insult or deficient tear production might belong to one of the following immunophenotype patterns: I--the number of T-cells that expressed CD3(+) CD8(+) was higher than the number of cells that expressed CD3(+)CD4(+). II--the number of cells CD3(+)C4(+) was higher than the number of cells CD3(+)CD8(+) and in this case a higher number of cells that expressed CD19 were identified. III--Proximity of values of the cells that expressed CD3(+)CD4(+) and CD3(+)CD8(+). These data might suggest that the number of lymphocyte T cells alone should not be considered a direct indicator of the presence of an immune-based inflammation. Besides, a particular population of T-cells does not indicate a particular inflammatory state. The morphological study of the third eyelid revealed a rather uncommon angioarchitecture. The artery that irrigates the eyelid crosses almost the entire length of this structure to achieve its free border, and only then, ramificates deeply towards an inner segmental level. This spatial microvascular arrangement probably results from an adaptation to the fact that the third eyelid, in the medial cantus of the eye, is inwardly compressed into a tiny space. Efficient vascularization is achieved by allowing the first ramifications of the third eyelid artery to run straight to the top. Accini secretor cells of the third eyelid show a mucin content while tubuloacinar cells are mainly serous.

Immunohistochemical distinction between preclinical bovine spongiform encephalopathy and scrapie infection in sheep.

Sheep are susceptible experimentally to bovine spongiform encephalopathy (BSE), the clinical signs being indistinguishable from those of scrapie. Because of the possibility of natural ovine BSE infection, laboratory tests are needed to distinguish between scrapie and BSE infection. The objectives of this study were to determine whether (1) PrPSc accumulates in biopsy samples of the tonsil or third eyelid, or both, of BSE-infected sheep before the appearance of clinical disease, and (2) such samples from BSE- and scrapie-infected sheep differ in respect of PrPSc accumulations. Homozygous ARQ sheep (n = 10) were dosed orally at 4-5 months of age with a brain homogenate from BSE-infected cattle. Third eyelid and tonsillar biopsy samples were taken at < or = 6 monthly intervals post-infection and examined immunohistochemically for PrPSc. Third eyelid protuberances were difficult to identify, resulting in many unsuitable samples; however, third eyelid samples shown to contain lymphoid follicles were invariably negative for PrPSc. In contrast, tonsillar biopsy samples became positive for PrPSc from 11 to 20 months post-infection. Consistent differences in the morphology of PrPSc granules in tingible body macrophages (TBMs) between BSE- and scrapie-infected sheep were detected with anti-peptide antibodies directed towards amino acids 93-106 of the ovine prion protein: thus, PrPSc appeared as single granules in TBMs of tonsillar sections from BSE-infected sheep, whereas clusters of PrPSc granules were observed within TBMs in the tonsils of scrapie-infected sheep. In contrast, antibodies against epitopes situated N- and C-terminally from the 93-106 region of the ovine prion protein revealed no differences between BSE- and scrapie-infected sheep in terms of PrPSc granules in TBMs.

IgA and secretory component (SC) in the third eyelid of domestic animals: a comparative study.

OBJECTIVE: The third eyelid of domestic animals is important for the production and distribution of tears, in removing ocular debris and in protection of the globe, and has significant immunologic functions. Although it is known that tears contain antibodies of the immunoglobulin A (IgA) isotype which are produced mainly by plasma cells of the lacrimal gland, very little is known about the antibody repertoires in the third eyelid of domestic animals. To assess whether IgA is derived from local synthesis, we analyzed the location of IgA-producing cells and the cellular distribution of secretory component (SC) in the third eyelid of domestic animals in a comparative study. ANIMAL STUDIED: A total of 83 third eyelids of dogs, cats, pigs, cows, sheep, goats and horses were investigated in the course of this study. PROCEDURES: Third eyelids were obtained immediately after death, cut length-wise, fixed overnight and processed for immunohistochemical detection of IgA and SC by the ABC technique. RESULTS: The results show that IgA-producing plasma cells are densely populated in subepithelial spaces of the surface epithelium as well as in the nictitating gland in a species-specific manner. In contrast, the SC could be demonstrated exclusively in glandular acinar and ductal epithelial cells and in different cell types of the surface epithelium, preferentially located on the bulbar side of the nictitating membrane. CONCLUSION: It is suggested that most of the SC is locally produced by resident plasma cells and subsequently transferred through the surface epithelium and glandular duct cells by transcytosis. This indicates that the third eyelid is an important member of the secretory immune system in domestic animals.

Histology, histochemistry and fine structure of the lacrimal and nictitans gland in the South American armadillo Chaetophractus villosus (Xenarthra, Mammalia).

The anatomical, histological, histochemical and ultrastructural characteristics of the lacrimal gland (LG) and nictitans gland (NG) of the armadillo Chaetophractus villosus were described. The histochemical and histological features of both glands in male and female adult animals were compared. The tissues were processed with conventional techniques for light and transmission electron microscopy. Fixed specimens were submitted to a battery of tests for glycans, glycosaminglycans, glycoconjugates, proteins, and lipids. The LG of the armadillo may be considered within the set of glandulae lacrimales superior in which primates, carnivores, perisodactyls and artiodactyls are included. The localization of the NG was similar to that of other mammals. Lacrimal and NG were histologically and histochemically identical. The secretory endpieces consisted of three cell types: (1) Mucous cells (MC) with different types of mucous secretory granules with neutral and sialic acid-containing glycoconjugates (GCs). (2)Seromucous cells (SMC) showing a variety of moderately electron dense secretory granules with flocculent material with carboxylated acidic, neutral, and sialic acid-containing GCs. Intercellular canaliculi with junctional complexes and basolateral intercellular spaces were frequent. (3) Serous cells (SC) with electron dense secretory granules. Histochemically, they showed the strongest reaction for proteins and neutral, weakly acid and carboxylated acidic GCs. The epithelium of the intra- and inter-lobular excretory ducts showed secretory activity, junctional complexes, and wide basolateral intercellular spaces with lateral folds. The endpieces and ducts were surrounded by myoepithelial cells. The stroma was characterized by fenestrated endothelium, unmyelinated axons, and abundant plasma cells. MC, SMC, and the duct system were richly innervated by hypolemmal nerve terminals.

Effect of a reserpine-like agent on the release and metabolism of [3H]NA in cell bodies and terminals.

1. The reserpine-like agent, Ro4-1284 (2-hydroxy-2ethyl-3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro- 11b-[H] benzo (a)quinolizine) releases [3H]noradrenaline ([3H]NA) from prelabelled superior cervical ganglion (cell bodies) and nictitating membrane (nerve endings) of the cat. 2. The potency of Ro 4-1284 29.0 microM was higher in the cell bodies than in the nerve endings. 3. In both tissues, exposure to the reserpine-like agent Ro 4-1284 induced a selective increase in the spontaneous outflow of [3H]DOPEG, while the [3H]OMDA metabolites to the release induced by Ro 4-1284 was very small. 4. The desamination is the preferential way of the metabolic inactivation of the [3H]NA released by the reserpine-like agent in both parts of the noradrenergic neuron.

In vitro release of [3H]noradrenaline by tyramine from the superior cervical ganglion and in the nictitating membrane of the cat.

1. The release and the metabolism of [3H]noradrenaline ([3H]NA) induced by tyramine was studied in the superior cervical ganglion (cell bodies) and in the nictitating membrane (nerve endings) of the cat. 2. Exposure of the ganglia to 58.0 and 174.0 microM tyramine resulted in the release of 13.7 and 11.8% respectively of the total tissue radioactivity. In the nictitating membrane, the fractional release of radioactivity was directly proportional to the concentration of tyramine (5.8, 58.0 and 174.0 microM). 3. In ganglia [3H]DOPEG accounted for 55.8% of the radioactivity released by 58.0 microM tyramine and only 10.5% of the radioactivity was unmetabolized NA. In presence of 174.0 microM tyramine, [3H]NA increased to 28.0% of the total radioactivity and [3H]DOPEG and [3H]OMDA decreased to 45.3 and 18.9% respectively. 4. In the nerve endings, the contribution of [3H]NA, [3H]DOMA and [3H]NMN increased with the concentration of tyramine while [3H]DOPEG decreased. 5. The deamination is the first step of the metabolic inactivation of [3H]NA induced by tyramine in the cell body of the postganglionic adrenergic neuron while in the nerve endings [3H]NA is preferentially O-methylated.

Classical conditioning selectively increases AMPA receptor binding in rabbit hippocampus.

The NMDA and AMPA receptors have been shown to play critical roles in various forms of synaptic plasticity (learning and memory, long-term potentiation). The present study investigated the involvement of these two receptors in a well-characterized classical conditioning paradigm. Following classical conditioning of the rabbit nictitating membrane the binding properties of these two subclasses of excitatory amino acid transmitter receptors were analyzed in dorsal hippocampi by quantitative autoradiography. [3H] TCP and [3H] AMPA were used to identify the NMDA and AMPA receptors, respectively. The binding of [3H]TCP to the NMDA receptor remained unchanged in all the experimental groups tested. Paired presentations of the conditioned and unconditioned stimuli resulted in increased [3H] AMPA binding to the AMPA receptor in several subfields of the hippocampus, while unpaired presentations had no significant effects. The increase in binding was due to an increased affinity of the low-affinity component of the AMPA receptor. The results support the hypothesis that changes in glutamate receptors participate in the synaptic plasticity involved in certain forms of learning.

Benzodiazepines decrease the release of [3H]noradrenaline and of [3H]acetylcholine in the cat superior cervical ganglion.

The effects of two benzodiazepines, diazepam and clonazepam, were studied on the release of [3H]noradrenaline and of [3H]acetylcholine elicited by preganglionic stimulation of the cat isolated superior cervical ganglion and on the contractile responses evoked by either nerve stimulation or exogenous noradrenaline in the cat isolated nictitating membrane. Both 10 microM diazepam and 10 microM clonazepam reduced by approximately 50% the release of [3H]noradrenaline and of [3H]acetylcholine in the cat ganglion whereas they did not modify the contractile responses in the nictitating membrane. It is concluded that benzodiazepines are also peripheral neuroactive agents and that they exhibit a tissue-selective action within the same animal species.

An in vivo model for dissociating alpha 2-and DA2-adrenoceptor activity in an ocular adnexa: utility of the cat nictitating membrane preparation.

Recent reports from this laboratory indicate that dopamine (DA2) agonists can lower intraocular pressure (IOP) in rabbits, monkeys and rats. This communication suggests that the cat nictitating membrane (CNM) preparation is a useful model for localizing the site(s) and mechanism(s) of action of dopamine (DA2) agonists and that this model can be used to discriminate between alpha 2 and DA2 activity. This in vivo preparation consists of indirect (neuronal)- and direct (agonist)-induced activation of the CNM by: 1) pre- and postganglionic stimulation of sympathetic nerves and 2) injection of norepinephrine (NEPI) into the external carotid artery (i.a.). A variety of DA2-agonists (ergolines, azepines, aminotetralins, aporphines) can cause dose-dependent depression of neuronally mediated contractions with minimal effects on contractions elicited by NEPI i.a. Characteristically, DA2-agonists depressed contractions elicited by low frequency (2 & 4 Hz) stimulation more than high frequency (6 & 8 Hz) stimulation. For example, the inhibitory effects of an ergoline derivative LY141865 on neuronally mediated contractions of the CNM could be reversed (or prevented) by relatively selective DA2 antagonists, such as sulpiride and domperidone, but not by relatively selective alpha 2-antagonists, such as yohimbine and rauwolscine. Interestingly, alpha 2-adrenoceptor agonists, such as clonidine and xylazine, caused less depression of nerve-stimulated responses than did DA2-agonists at the same dose. This modest suppression by alpha 2-agonists could be inhibited by yohimbine but not by sulpiride or domperidone. These results with the CNM document the presence of DA2 receptors on sympathetic neurons innervating an ocular adnexa and demonstrate a model for dissociating DA2 from alpha 2 activity.

Drug reservoirs in topical therapy.

The nictitating membrane, corneal epithelium, and corneal stroma were investigated as drug reservoirs. A hydrophilic drug, D,L-epinephrine HCl, or a lipophilic drug, chloramphenicol, was applied topically to rabbit eyes. Tissue levels of radioactive drug-plus-metabolites and unmetabolized epinephrine were assayed up to 24 hrs later. On a per-mg-tissue basis, concentrations of epinephrine-plus-metabolites in the stroma-endothelium were similar to or higher than those in the epithelium. The percentages of radioactivity representing unmetabolized epinephrine in the stroma-endothelium were found to be similar to or higher than those in the epithelium. On a per-mg-tissue basis, concentrations of chloramphenicol-plus-metabolites were significantly higher in the epithelium than in the stroma-endothelium during the first 130 min after drug application. While the physical properties of these drugs determined whether a higher concentration was found in the epithelium or stroma-endothelium, the ninefold greater mass of the stroma-endothelium made it the major drug reservoir on a per-whole-tissue basis. The presence or absence of a nictitating membrane had little effect on the level of either drug in the epithelium, stromal-endothelium, or aqueous humor.

Ionic mechanisms involved in the release of 3H-norepinephrine from the cat superior cervical ganglion.

It has previously been reported that in the isolated cat superior cervical ganglion (SCG) labeled with tritiated norepinephrine (3H-NE), the stimulation of the preganglionic trunk at 10 Hz as well as the exposure to 100 microM exogenous acetylcholine (ACh), produced a Ca++-dependent release of 3H-NE. The present results show that a Ca++-dependent release of 3H-NE was produced also by exposure to either 50 microM veratridine or 60 mM KCl. Tetrodotoxin (0.5 microM) abolished the release of 3H-NE induced by preganglionic stimulation, ACh and veratridine but did not modify the release evoked by KCl. The metabolic distribution of the radioactivity released by the different depolarizing stimuli showed that the 3H-NE was collected mainly unmetabolized. In the cat SCG neither the release of 3H-NE evoked by KCl nor the endogenous content of NE was modified by pretreatment with 6-OH-dopamine (6-OH-DA). On the other hand, this chemical sympathectomy depleted the endogenous content of NE in the cat nictitating membrane, whose nerve terminals arise from the SCG. The data presented suggest that the depolarization-coupled release of NE from the cat SCG involves structures that are different to nerve terminals and that contain Na+ channels as well as Ca++ channels.

In vitro release of 3H-noradrenaline by amphetamine from the superior cervical ganglion of the cat.

The release and metabolism of 3H-noradrenaline (3H-NA) produced by d-amphetamine was studied in the superior cervical ganglion of the cat (cell bodies) and in the nictitating membrane (nerve endings). Exposure of the nictitating membrane to 10 microM d-amphetamine, resulted in the release of 5.1% of the total radioactivity. This was mainly collected as 3H-normetanephrine (3H-NMN) and as 3H-NA; 3H-3,4-dihydroxyphenylglycol (3H-DOPEG) did not contribute to the drug-induced outflow of radioactivity. In contrast, exposure of ganglionic cell bodies to 10 microM d-amphetamine for 10 min released only 1.74% of the total tissue radioactivity and 3H-DOPEG represented the most important fraction of the released radioactivity. When ganglia were exposed to 30 microM d-amphetamine, the radioactivity released was 5.2%; the proportion of 3H-NA and 3H-NMN increased and 3H-DOPEG was reduced. These results show that: a) d-amphetamine releases 3H-NA from prelabeled cell bodies and nerve endings; b) the potency of d-amphetamine was higher in nerve terminals than in cell bodies, and c) at low concentrations of d-amphetamine, the metabolism of the released neurotransmitter differed between both parts of the adrenergic neuron.

Pharmacological differentiation of presynaptic inhibitory alpha-adrenoceptors and opiate receptors in the cat nictitating membrane.

1 The action of morphine, naturally occurring and synthetic opiate peptides on [3H]-noradrenaline release induced by nerve stimulation was studied in the isolated nerve muscle preparation of the cat nictitating membrane under experimental conditions in which the alpha-presynaptic receptors were blocked by phentolamine 1 microM. 2 Morphine and the naturally occurring peptides: [Met5]-enkephalin, [Leu5]-enkephalin and beta-endorphin reduced 3H-transmitter overflow and responses to nerve stimulation from the cat nictitating membrane, effects which were completely antagonized by naloxone 0.3 microM. The relative order of potency for the inhibition of the stimulation-induced 3H-transmitter overflow at the level of the IC50 (microM) was as follows: [Met5]-enkephalin (0.020 microM) greater than or equal to [Leu5]-enkephalin (0.036 microM) > morphine (0.3 microM) > beta-endorphin (1 microM). 3 The synthetic opiate pentapeptides: BW 180 C (Tyr-D-Ala-Gly-Phe-D-Leu), and BW834 C (Tyr-D-Ala-Gly-pClPhe-DLeu), which are resistant to enzymatic degradation were more potent than the enkephalins in reducing the stimulation-evoked transmitter overflow from the cat nictitating membrane. On the other hand, the tetrapeptide BW832 C, which lacks the D-leucine terminal of BW180 C l was less potent than the enkephalins in inhibiting neurotransmission. 4 In the presence of phenoxybenzamine 1 microM, 3H-transmitter overflow was increased 8 fold and the inhibition of neurotransmission by methionine-enkephalin was not affected. Exposure to phenoxybenzamine 10 microM increased [3H]-noradrenaline overflow 15 fold and antagonized the effects of methionine enkephalin on transmitter release. 5 In the cat nictitating membrane the inhibitory presynaptic opiate receptors are different from the presynaptic alpha-autoreceptors which regulate the release of noradrenaline elicited by nerve depolarization through a negative feed-back mechanism.

Release of radioactive purines from cat nictitating membrane labeled with 3H-adenine.

Cat nictitating membranes were incubated with 1-2 x 10(-7) M 3H-adenine or 3H-adenosine for 1 h. A tissuebath ratio of about 15 was found for both compounds in intact and denervated membranes. In intact nictitating membranes sympathetic nerve stimulation (4 Hz, 5 min) caused a net release of purines (0.66 +/- 17% of the tissue content), which was reduced by alpha-blockade. Noradrenaline (1-3 microM) or tyramine 60 microM), which produced the same contractile response as did nerve stimulation, increased purine release to the same extent as did nerve stimulation. The effect of either agent was reduced or abolished by phentolamine. Purine release could also be induced by acetylcholine and ATP. This release was not altered after surgical denervation. There was an excellent correlation between the contractile response and the purine release induced by nerve stimulation, noradrenaline, tyramine and acetylcholine. However, ATP caused a larger release of 3H-purines than expected from the contractile responses, possibly indicating displacement. The results indicate that most if not all of the 3H-purines released by nerve stimulation in the cat nictitating membrane are derived from postjunctional elements.