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PubMed1991

The direct contractile effect of gastrin releasing peptide on isolated gastric smooth muscle cells of the guinea pig.

Smooth muscle cells isolated from the gastric muscle layers of the guinea pig were used to determine whether gastrin releasing peptide (GRP) can cause contraction by exerting a direct action on muscle cells. In addition, the inhibitory effect of 8-( N,N-diethylamino )-octyl-3,4,5-trimethoxybenzoate hydrochloride ( TMB-8 ), an inhibitor of intracellular Ca2+ release, and verapamil, a Ca2+ channel blocker, on the GRP-induced contraction of gastric smooth muscle cells were examined. GRP elicited a contractile response of gastric muscle cells in a dose-dependent manner. The ED50 was 13 pM. TMB-8 significantly inhibited the contractile effect of GRP in gastric muscle cells. These results demonstrate the direct action of GRP on the gastric smooth muscle cells of the guinea pig, and the importance of Ca(2+)-release from intracellular calcium, stores in the contractile response to GRP.

Animals↗
PubMed1992

Direct contractile effect of motilin on isolated smooth muscle cells of guinea pig small intestine.

We examined the direct effect of motilin on longitudinal and circular smooth muscle cells isolated from the guinea pig small intestine. In addition, the effects of 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxy-benzoate hydrochloride (TMB-8, an inhibitor of intracellular Ca(2+)-release), verapamil (a voltage-dependent Ca(2+)-channel blocker), and removal of extracellular Ca2+ were investigated to evaluate the role of intracellular Ca2+ stores and extracellular Ca2+ on the muscle contraction induced by motilin. The effects of atropine (a muscarinic receptor antagonist), spantide (a substance P receptor antagonist) and loxiglumide (a CCK-receptor antagonist) were also examined to determine whether the motilin-induced contraction was independent of those receptors. Motilin induced a contraction of the longitudinal and circular smooth muscle cells in a dose-dependent manner with the maximal effect attained after 30 seconds of incubation. The ED50 values were 0.3 nM and 0.05 nM, respectively. TMB-8 suppressed completely the motilin-induced contraction of both types of smooth muscle cells. Verapamil had only a slight suppressive effect. Removal of extracellular Ca2+ did not have any significant influence on motilin-induced contraction. The contractile response to motilin was not affected by atropine, spantide or loxiglumide. Our findings showed that:1) motilin has a direct contractile effect on both longitudinal and circular smooth muscle cells; 2) this contractile effect is not evoked via muscarinic, substance P or CCK receptors, and 3) the intracellular release of Ca2+ plays an important role in the contractile response to motilin on both types of smooth muscle cells.

Animals↗
PubMed1985

Ca2+-dependent modulation of renin release from isolated glomeruli: apparent independence from alterations in cGMP.

This study examined the role of cGMP in the control of renin release from isolated rat glomeruli. An inverse correlation between renin release and cGMP content of isolated glomeruli was found under several conditions of incubation. Thus, incubation of isolated glomeruli in Ca2+-free media containing EGTA, or the addition of the Ca2+ and calmodulin (CaM) antagonists trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride (W-7), or (8-N, N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCI (TMB-8) to glomeruli incubated in Ca2+ replete buffer lowers cGMP and stimulates renin release. These same incubation conditions enhance the release of renin induced by isoproterenol (DBcAMP) in isolated glomeruli. By contrast, raising media K+ to 60 mmol/L, or the incubation of glomeruli with angiotensin II (A-II) or ouabain--all of which are thought to increase intracellular Ca2+--increased glomerular cGMP and suppressed basal glomerular renin release and the increases in renin release induced by isoproterenol (DBcAMP). However, neither exogenous DBcAMP nor nitroprusside, an agent that increased the endogenous cGMP, content of glomeruli mimicked the suppressive effects of high K+, A-II, or ouabain on renin release. Moreover, DBcGMP and nitroprusside also failed to reverse the stimulatory effects of Ca2+ deprivation, TFP, W-7, or TMB-8 on glomerular renin release, even though nitroprusside clearly enhanced cGMP under these conditions of incubation. The results suggest that changes in glomerular cGMP and renin release occur concomitantly in response to alterations in glomerular Ca2+ homeostasis, but that cGMP does not mediate the changes in glomerular renin release.

1-Methyl-3-isobutylxanthine↗
PubMed1991

An investigation of the sensitivity of the ouabain-insensitive sodium efflux in single barnacle muscle fibers to pentachlorophenol.

The aim of the present work was to explore the possibility that pentachlorophenol (PCP) influences the behavior of the resting Na efflux in single muscle fibers from the barnacle, Balanus nubilus. It is shown here that PCP causes a transitory rise in the Na efflux in both unpoisoned and ouabain-poisoned fibers and that the response is dose-dependent, the minimal effective concentration in ouabain treated fibers being less than 10(-6) M. The efficacy of PCP is significantly greater than that of 2,3,4-trichlorophenol. 2,3-Dichlorophenol is ineffective. This is also the case with phenol. The magnitude of the response to PCP is a function of external pH. Lowering pHe increases the response. The response has an absolute requirement for external Ca2+ and is a sigmoidal function of external Ca2+ concentration. Since treatment of these fibers with PCP in high concentration leads to prompt contraction, experiments were designed to determine whether the observed rise in ouabain-insensitive Na efflux is due to a fall in myoplasmic pCa and whether trigger Ca2+ originates from the bathing medium. The results obtained show that prior injection of ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxyethane-N,N,N',N'-tetraacetic acid (BAPTA) leads to a drastic reduction in the response to PCP. They also show that prior external application of verapamil or devapamil stops the response to PCP from occurring. Both Cd2+ and Co2+ are also effective but only temporarily. Last, the effects of ryanodine and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) were tested, since the former is known to block the sarcoplasmic reticulum Ca2+ release channel, and the latter to impair the action of agents known to release Ca2+ from internal depots. Both ryanodine and TMB-8 are found to reduce the response to PCP. Taken together, these observations support the hypothesis that PCP stimulates the ouabain-insensitive Na efflux by increasing the internal free Ca2+ and that the increase in internal Ca2+ is due to the entry of trigger Ca2+ from the outside via Ca2+ channels, as well as release of Ca2+ by the sarcoplasmic reticulum via its channel. They also indicate that the efficacy of PCP depends on the 5 Cl atoms present in its aromatic ring and pHe.

Animals↗
PubMed1986

The effect of calcium antagonists on histamine and leukotriene-induced tracheal microvascular permeability in the guinea pig.

The effects of several calcium antagonists, i.e., nifedipine, verapamil and 8-[N,N-diethylamino]-octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8), were evaluated in situ on agonist-induced increases in permeability of the airway microvasculature in anesthetized guinea pigs. Vascular permeability was measured as tracheal extravascular albumin content by using 125I-bovine serum albumin and the utilization of 51Cr labelled-erythrocytes to correct for blood volume. Intratracheal injections of histamine (1, 10 and 100 micrograms) or leukotriene (LT) D4 (1, 10 and 100 ng) produced dose-dependent increases in extravasated radiolabelled albumin in the trachea. Although histamine produced a greater maximal response than LTD4, the latter provocation was ten times more potent than the former. Nifedipine, a dihydropyridine calcium slow channel blocker, exhibited dose-dependent (30, 100 and 300 micrograms/kg) inhibitory activity against histamine-induced increases in extravascular albumin, while another calcium slow channel blocker, verapamil (100, 300 and 1000 micrograms/kg), exhibited much less activity. TMB-8, a purported intracellular calcium antagonist (1 and 10 mg/kg), was observed to have some inhibitory activity versus histamine. Similar doses of all three calcium antagonists failed to significantly inhibit increases in tracheal microvascular permeability evoked by LTD4. These results suggest that differences in mediator-induced microvascular permeability in the guinea pig trachea are evident depending upon the agonist selected and the pool of calcium utilized.

Animals↗
PubMed1982

The role of calcium in macrophage chemotaxis to the phorbol ester tumor promoter TPA.

The significance of the macrophage in the inflammatory response that occurs concurrently with phorbol ester induced tumor promotion has not yet been determined. Biologically active phorbol ester tumor promoters modify several functional responses of macrophages including chemotaxis, cytotoxicity, secretion and prostaglandin synthesis and release. The present study examines calcium metabolism as a possible underlying biochemical mechanism through which 12-0-tetradecanoyl-phorbol-13-acetate (TPA) exerts its effects on macrophage chemotaxis. The chemotaxis of mouse resident peritoneal macrophages was evaluated in the presence of pharmacological agents known to alter cellular calcium metabolism. The calcium ionophore A23187 in microM concentrations enhanced macrophage chemotaxis to TPA by approximately 41%. This enhancement was dependent on the presence of extracellular calcium. TPA-induced chemotaxis was also enhanced by the histological dye ruthenium red (RR), an agent known to modify mitochondrial calcium fluxes and calcium-dependent neuronal transmission. Ruthenium red (0.1 and 1.0 microM) produced a maximal stimulation of macrophage chemotaxis to TPA of approximately 62%. An intracellular calcium antagonist, 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) inhibited macrophage chemotaxis to TPA in a dose related fashion (1.0 to 100 microM). Varying extracellular calcium concentrations (0-3.6 mM) had no effect on macrophage chemotaxis in response to TPA. In drug combination studies neither A23187 nor RR was able to overcome the inhibitory effects of TMB-8 on macrophage chemotaxis to TPA. These results indicate that intracellular calcium metabolism may be playing a significant role in modulating TPA's effect on macrophage chemotaxis, while extracellular calcium may be of little import. A possible mode of TPA's effect on the macrophage via mobilization of calcium from cellular storage sites is discussed.

Animals↗
PubMed1985

Alterations in calcium metabolism in phorbol ester-treated mouse peritoneal macrophages.

Phorbol 12-myristate 13-acetate (PMA)-treated macrophages exhibited a two-fold increase in the rate of 45Ca++ efflux and over a three-fold increase in the size of the exchangeable calcium pool, resulting in almost a seven-fold increase in the slow phase of calcium efflux. The calcium antagonist 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) by itself did not affect calcium efflux in macrophages; but abolished the PMA-induced increase in the rate of calcium efflux. The divalent cationphore A23187 increased the rate constant of the fast phase of calcium efflux two-fold when applied alone or when applied with PMA. These effects might be linked to ionophore enhancement and TMB-8 inhibition of PMA-induced macrophage chemotaxis and spreading (previously reported in Cell Calcium 3:503-514 and Cancer Research 43:3385-3391). No change in calcium efflux was observed if cells were exposed to PMA only during the efflux experiment suggesting that a prolonged exposure to PMA is required to elicit changes in calcium flux. Increased 45Ca++ remained in treated cells at each time point perhaps reflecting the PMA-induced increase in exchangeable calcium.

Animals↗
PubMed1981

Anterograde tracing of horseradish peroxidase (HRP) with the electron microscope using the tetramethylbenzidine reaction.

A technique is described for anterograde horseradish peroxidase (HRP) tracing with the electron microscope using tetramethylbenzidine (TMB) histochemistry. Following HRP injection into the primary visual cortex or one eye of rabbits the superior colliculus was studied with the light and electron microscope in adjacent sections. In target preparations from the colliculus examined with the electron microscope labelled axons and axon terminals were found. The TMB reaction product was identified as crystal like electron-dense structures. In control sections from the opposite colliculus the neuropil was free from label.

Afferent Pathways↗
PubMed1983

Improved visualization of neurons labeled with horseradish peroxidase: silver-intensification of the pyrocatechol/p-phenylenediamine reaction product.

A silver intensification procedure suitable for use with pyrocatechol/p-phenylenediamine (PC-PPD) product of the horseradish peroxidase (HRP) reaction is described. Qualitative and quantitative results from retrogradely labeled neurons in the cat cortex after thalamic injection of HRP demonstrate an increase of the intensity of labeling and in the number of darkly labeled cells after the intensification procedure. In both the non-intensified and the intensified PC-PPD reacted tissue the sensitivity was comparable to that of TMB-treated material. The ratio of lightly to darkly labeled neurons was very similar in intensified PC-PPD and TMB material, suggesting that the lightly labeled cells may have fewer terminals present at the level of the injected target.

Animals↗
PubMed1989

A triple-labeling method: HRP anterograde tract tracing combined with double immunofluorescent cell staining in developing neural tissue of the rat.

In this paper we describe a triple-labeling technique on a single section of developing nervous tissue. This labelling consists of a histochemical visualization of anterogradely transported horseradish peroxidase (HRP), within outgrowing rat corticospinal tract fibers and a double immunofluorescence labelling against glial fibrillary acidic protein (GFAP) and vimentin, being specific markers for astrocytes and their precursors, respectively. For the visualization of the anterogradely transported HRP, tetramethylbenzidine (TMB) is used as a chromogen with ammonium heptamolybdate (AHM) as a stabilizer followed by an additional diaminobenzidine-cobalt incubation to prevent washing out the HRP-TMB-AHM reaction product during the subsequent immunohistochemistry. Thereafter, double immunofluorescence labelling is carried out on the same section by adding simultaneously polyclonal anti-GFAP and monoclonal anti-vimentin which are then visualized with antisera conjugated with tetramethylrhodamine-isothiocynate (TRITC) and fluorescein-isothiocyanate (FITC) respectively as markers. Since the above mentioned visualization of the anterogradely transported HRP does not affect the immunofluorescence labelling, the methods enables within a single cryosection of developing neural tissue, the localization and identification of both outgrowing axons and their adjacent differentiating glial cells. In addition the method can easily be adapted for triple labelling of HRP tract tracing in conjunction with double immunofluorescence histochemistry of neurotransmitters.

Animals↗
PubMed1985

Central actions of calcitonin on body temperature and intestinal motility in rats: evidence for different mediations.

The effects of intracerebroventricular (i.c.v.) administration of calcitonin and PGE2 on intestinal motility and body temperature were examined in conscious rats chronically fitted with intraparietal electrodes in the small intestine, a cannula in a cerebral lateral ventricle and a subcutaneous thermistor probe. Both calcitonin and PGE2 restored the fasted pattern of intestinal motility in fed rats and induced an increase in body temperature. Indomethacin, an inhibitor of the cyclooxygenase with calcium antagonistic properties, and TMB-8, an intracellular calcium antagonist, blocked the effects of calcitonin on intestinal motility and body temperature. Piroxicam, an inhibitor of the cyclooxygenase which does not affect calcium uptake blocked the thermic but not the intestinal effects of calcitonin. TMB-8 but not indomethacin or piroxicam partially blocked the effects of PGE2 on both intestinal motility and body temperature. It is concluded that the central hyperthermic effect of calcitonin is mediated through the formation and the release of prostaglandins whereas the central action of calcitonin on digestive motility results from intracerebral effects on calcium fluxes.

Animals↗
PubMed1982

The effects of platelet secretion inhibitors on protein phosphorylation.

Protein phosphorylation was investigated in human platelets after stimulation to secretion by thrombin. After stimulation by thrombin at 4 degrees C (in which secretion is inhibited), phosphorylations of the 80, 56, and 38 kDa polypeptides and dephosphorylation of the 67 kDa phosphopeptide eventually occurred. The phosphorylations of the 27 and 20 kDa polypeptides remained inhibited until the temperature was increased to 37 degree C, which also resulted in secretion. Various stimulants and inhibitors of platelet function were used to characterize individual protein phosphorylations. The divalent-cation ionophore, A23187, induced the phosphorylations (or dephosphorylation) of the same proteins as thrombin with the exception of the 80 kDa protein, which remained incompletely phosphorylated. The intracellular calcium antagonist, TMB-8, inhibited thrombin-stimulated secretion and phosphorylation of all the polypeptides except the 80 kDa protein. The dephosphorylation of the 67 kDa phosphoprotein was not affected by TMB-8. Incubation of platelets with prostaglandin E1 and isobutylmethylxanthine inhibited thrombin-stimulated secretion and the phosphorylation of the 38 and 20 kDa protein and increased the phosphorylation of the 67 and 27 kDa phosphoproteins. These observations may be used to correlate protein phosphorylation with secretion, suggesting a possible sequence of intracellular events that mediate thrombin-stimulated secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Superoxide production of human polymorphonuclear leukocytes stimulated by leukotriene B4.

Leukotriene B4 stimulated a transient production of superoxide anions (O2-) by human polymorphonuclear leukocytes which continued for only about 1 min. The production was dependent on Ca2+ in the suspending medium and no production was observed without the addition of calcium. The concentrations of leukotriene B4 and calcium for the half-maximal production were about 1 microM and 200 microM, respectively. 8-(N,N,-Diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8), an intracellular calcium antagonist, did not inhibit the O2- production stimulated by leukotriene B4 in the presence of calcium, while N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, did. When leukotriene B4 was added to the cells treated with cytochalasin B, the production of O2- was biphasic: an initial rapid phase, followed by a slow one. The slow phase was also dependent on Ca2+ concentrations but it could be induced even without the addition of Ca2+ to the medium. The cells treated with both cytochalasin B and TMB-8 in Ca2+-free medium showed a negligible production of superoxide on addition of leukotriene B4, but the production appeared upon addition of CaCl2. These findings suggest that the superoxide production stimulated by leukotriene B4 is associated with the influx of Ca2+.

Animals↗
PubMed1985

Differential effects of extracellular calcium removal and nonspecific effects of Ca2+ antagonists on acid secretory activity in isolated gastric glands.

The role of extracellular calcium in the action of the secretagogues, carbachol, histamine and forskolin, on parietal cell HCl secretion was investigated using glands isolated from rabbit gastric mucosa. Omission of calcium from the cellular incubation medium and chelation of a major portion of contaminating calcium with EGTA resulted in a disappearance of the initial transient response to carbachol (as measured by uptake of the weak base, amino[14C]pyrine), but the sustained response to carbachol persisted. Neither histamine nor forskolin-stimulated increase in amino[14C]pyrine uptake were affected by omission of extracellular calcium. Furthermore, the potentiating interactions between histamine and carbachol and between forskolin and carbachol appeared to occur independent of extracellular calcium. Attempts to assess the contribution of intracellular calcium to secretory activity using the Ca2+ antagonists, verapamil, nifedipine, nicardipine and lanthanum, and the putative intracellular Ca2+ antogonist, TMB-8 (3,4,5-trimethyloxybenzoic acid 8-(diethyl-amino)-octyl ester) were unsuccessful. Nifedipine had no effect on secretagogue stimulated amino[14C]pyrine accumulation even at concentration well above the pA2 reported for excitable tissues. Verapamil, nicardipine, lanthanum and TMB-8 all appeared to have nonspecific inhibitory effects on amino [14C]pyrine uptake. From these results we conclude that: (1) parietal cell HCl secretion can occur independent of extracellular Ca2+; (2) influx of extracellular Ca2+ enhances the response to carbachol but has little influence on the secretory response initiated by cAMP-dependent secretagogues; and (3) parietal cell Ca2+ channels have a different molecular configuration than Ca2+ channels in excitable cells.

Aminopyrine↗
PubMed1992

Relationship between cytosolic Ca2+ concentration and amylase release in rat parotid acinar cells following muscarinic stimulation.

Carbachol (CCh), a muscarinic-cholinergic agonist, increased both cytosolic free calcium concentration ([Ca2+]i) and amylase release in rat parotid acinar cells or acini in a dose-dependent manner. Treatment of acinar cells with the intracellular Ca2+ antagonist, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), or the intracellular Ca2+ chelator, 1,2-bis(O-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA), strongly attenuated the increases in [Ca2+]i evoked by CCh, but amylase release from acini was not significantly suppressed by the treatment with TMB-8 or BAPTA. Low concentrations (0.02-0.5 microM) of ionomycin, a Ca2+ ionophore, caused increases in [Ca2+]i comparable to those induced by CCh, but the same concentrations had only a little effect on amylase release. The protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated amylase release in quantities similar to those induced by CCh, although TPA alone did not cause any change in [Ca2+]i. Combined addition of TPA and ionomycin potentiated only modestly amylase release stimulated by TPA alone. Staurosporine, a protein kinase C-inhibitor, similarly inhibited both the CCh- and TPA-induced amylase release. These results suggest that an increase in [Ca2+]i elicited by CCh does not play an essential role for inducing amylase release in rat parotid acini. Amylase release by muscarinic stimulation may be mediated mainly by activation of protein kinase C.

Alkaloids↗
PubMed1992

Rapid treatment of whole cells and RNA viruses for analysis of RNA by slot blot hybridization.

To avoid extensive manipulation for the purification of RNA from cells, several methods were evaluated for the direct release of RNA from influenza virus infected cells and supernatants using slot blot hybridization and non-radioactive probes. Treatment with an equal volume of 10 M aqueous guanidine hydrochloride produced the best hybridization signal. Less, but significant amounts of RNA were also released using the following treatments: dilute alkali (final concentration of 0.16 M NaOH) or 100 degrees C/5 min or RNA sample buffer containing formamide/formaldehyde, then heating at 65 degrees C/10 min. Despite the presence of large amounts of cell debris, RNA from guanidine hydrochloride treated whole cell extracts bound quantitatively to the positively charged nylon membranes. The sensitivity of RNA detection when whole cell extracts treated with guanidine hydrochloride were probed with a digoxigenin labelled cDNA probe was similar to the detection of RNA in highly purified, protein free samples. Three positively charged membranes were tested (from Amersham, ICN and Boehringer Mannheim) using two alkaline phosphatase substrates, NBT-X phos, and a chemiluminescent substrate, 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoyloxy)-phenyl- 1-1,2-dioextane (AMPPD) and a peroxidase substrate, tetramethylbenzidine (TMB). The Boehringer Mannheim membrane had the highest sensitivity for the alkaline phosphatase substrates, but the peroxidase reaction with the TMB substrate was the most consistently sensitive, irrespective of which membrane was used. The ability to quantitatively detect RNA from whole cells without any purification will allow the rapid screening of large numbers of samples for specific RNA species in research or diagnostic laboratories.

Animals↗
PubMed1994

Role of calcium and calmodulin in the activation of murine peritoneal macrophages with cisplatin.

When fura-2/AM loaded peritoneal macrophages were treated with cisplatin, it resulted in a rapid increase in the intracellular free calcium. Calcium modulating agents, EGTA, nifedipine, and TMB-8, and the calmodulin antagonist W-7 inhibited cisplatin-induced tumoricidal activity of murine peritoneal macrophages. Supernatants collected from macrophages treated with cisplatin and EGTA, nifedipine, TMB-8 or W-7 demonstrated decreased tumor necrosis factor (TNF) and interleukin-1 (IL-1) activity in comparison to supernatants collected from macrophages treated with cisplatin alone. Similarly, TNF and IL-1 activity were significantly inhibited in paraformaldehyde (PFA)-fixed macrophages and freeze-thaw lysates of macrophages treated with cisplatin and different calcium and calmodulin-modulating agents. These results suggest that calcium and calmodulin are important in the cisplatin-mediated activation of murine peritoneal macrophages to tumoricidal state.

Animals↗
PubMed1994

Ultrastructural evidence for synaptic inputs of enkephalinergic nerve terminals to target neurons in the rat arcuate nucleus.

The morphological support of interactions between enkephalins and three systems--beta-endorphin (beta-END), tyrosine hydroxylase (TH), or neuropeptide Y (NPY)--well represented in the arcuate nucleus, was examined by using an electron microscopic double immunostaining combining two sensitive chromogens, diaminobenzidine (DAB) and tetramethylbenzidine (TMB). The first step consisted of visualizing Metenkephalinergic terminals with DAB reaction product, and the second one involved detecting the antigens TH, beta-END, and NPY in their respective neurons with TMB reaction product. Ultrastructural analysis revealed enkephalinergic terminals presynaptic to TH-immunopositive cells and dendrites, principally in the dorsal portion of the arcuate nucleus. Enkephalinergic nerve terminals also contacted synaptically ventrolaterally located beta-END-immunoreactive cells. In the ventromedial arcuate nucleus, few synaptic contacts were observed between enkephalinergic boutons and NPY neurons, which were principally in close apposition with glial processes. Enkephalin-immunoreactive synapses were more frequently seen on TH-immunopositive neurons. This TH neuronal group is known to correspond to the dopaminergic tuberoinfundibular neurons implicated in the control of reproductive functions. The pattern of distribution of the different synapses within the arcuate nucleus (TH dorsal, beta-END ventrolaterally; NPY ventromedially) suggests that enkephalins may play a role in the neuroendocrine regulation of gonadotropin and prolactin secretion. The results provide evidence that enkephalins, in the arcuate nucleus, exert a postsynaptic action on the beta-END cells in addition to the presynaptic regulation previously demonstrated in the mediobasal hypothalamus, related to beta-END release. Moreover, the arcuate nucleus is a site of intercellular relationships between enkephalins and dopamine and between enkephalins and other peptides such as NPY.

3,3'-Diaminobenzidine↗