[Photodynamic effect on cells of human adipose tissue in vitro].

Changes in optical properties of human adipose tissue cells after photodynamic exposure in vitro were found and investigated. Analysis of the kinetics of the process was realized by means of photomicrography of the object investigated. The statistical computer processing of digital photos obtained gave us an opportunity to estimate quantitatively the kinetics of photodynamic effect upon the tissue. Optical interpretation of the photos obtained indicates that the observed phenomenon corresponds to the partial lysis of adipose tissue cells without their complete destruction.

Opioid inhibition of dopamine release from nervous tissue of Mytilus edulis and Octopus bimaculatus.

Morphine and D-Ala2-Met-enkephalin as well as other opioids suppress potassium-stimulated release of 3H-labeled dopamine from neurons tissue of two marine invertebrates, Mytilus edulis and Octopus bimaculatus. Naloxone reverses the inhibitory effects in both species. Potassium-stimulated release of 3H-labeled serotonin is not altered by opioids. It is postulated that opiate receptors and their endogenous effectors play a prominent role in regulation of transmitter release in invertebrates.

A direct role of dopamine in the rat subthalamic nucleus and an adjacent intrapeduncular area.

The subthalamic nucleus, a clinically important component of the extrapyramidal motor system, and a lateral area extending into the peduncle contain catecholamine terminals and dopamine receptors coupled to adenylate cyclase. In addition, dopamine agonists administered in vivo enhance glucose utilization in the region. Thus, neuronal function in this region is directly affected by dopamine and dopaminergic drugs.

Binding sites for [3H]SCH 23390 in retina: properties and possible relationship to dopamine D1-receptors mediating stimulation of adenylate cyclase.

Bovine, rat and chick embryo retinal membranes contain high affinity, saturable and stereospecific binding sites for the selective dopamine D1-receptor antagonist, [3H]SCH 23390 (R-(+)-8-chloro-2,3,4,5-tetrahydro-3- methyl-5-phenyl-lH-3-benzazepin-7-ol). Saturation studies and Scatchard analyses showed a single class of [3H]SCH 23390 binding sites with Kd (apparent dissociation constant) values of 0.5-1.4 nM for the different species studied. A high ratio of specific to non-specific binding was found over a wide range of radioligand concentrations. The Bmax (binding site number) for [3H]SCH 23390 in calf retina was 307 +/- 38 fmol/mg protein, significantly greater than Bmax values previously obtained for binding of [3H]spiroperidol and [3H]ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene) to dopamine D2-receptors in calf retina. Relative affinities (Ki values) of dopamine antagonists for calf retinal [3H]SCH 23390 binding sites were similar to those reported for [3H]SCH 23390 binding in rat striatum and also were in general agreement with potencies for antagonism of retinal dopamine-stimulated adenylate cyclase. However, they differed markedly from the relative affinities for retinal D2-receptor sites. Additional data indicated that SCH 23390 did not bind significantly to retinal D2- or serotonergic receptors and had 30- to 80-fold less affinity for alpha 2-noradrenergic than for the [3H]SCH 23390 sites. Competition studies indicated a high degree of selectivity for dopamine agonist over other agonists for [3H]SCH 23390 binding sites with Ki values in the range expected for interaction with dopamine receptors mediating stimulation of adenylate cyclase. Affinity for dopamine was decreased in the presence of the GTP analogue, Gpp(NH)p. In the presence of sodium ions the affinities of dopamine agonists for [3H]SCH 23390 binding sites were markedly and selectively decreased; the sensitivity to dopamine for stimulation of adenylate cyclase activity was also decreased in the presence of sodium ions. Modulation by sodium ions was previously observed for D2- but not for a D1-receptor interaction. It is proposed that [3H]SCH 23390 binds to a unique class of receptors, most likely D1-receptors coupled to adenylate cyclase in retina. [3H]SCH 23390 provides a potent new tool for study of these receptors. In retina D1-receptors positively coupled to cyclase as well as D2- and other receptors that may be negatively coupled to cyclase, appear to be regulated by sodium ions as well as by guanine nucleotides.

Modulation of adenylate cyclase activity of mouse spinal cord-ganglion explants by opioids, serotonin and pertussis toxin.

Organotypic cultures of fetal mouse spinal cord-ganglion explants (2-4 weeks in vitro) contain forskolin-stimulated adenylate cyclase (AC) activity that is inhibited by levorphanol and other opioid agonists in a dose-dependent manner. Inhibition by levorphanol no longer occurs if sodium is omitted from the incubation and the levorphanol inhibition is blocked by the opioid antagonist, naloxone. These findings together with the ineffectiveness of dextrorphan indicate that the opioid inhibition of forskolin-stimulated AC is receptor mediated. Both the delta- and kappa-receptor subtypes appear to be involved since the selective delta-opioid agonist, [D-Pen2, D-Pen5]enkephalin, and the selective kappa-opioid agonist, t-3,4-dichloro-N-methyl-N[2-(1-pyrrolidinyl)cyclohexyl]-benzene acetamide (U-50,488H) are both effective at nanomolar concentrations. In contrast, the selective mu-opioid agonist, Tyr-D-Ala-Gly-N-MePhe-Gly-ol, has no significant effect even at micromolar concentrations. Both cord and ganglion components of the explants contain opioid-sensitive AC. Forskolin-stimulated AC of the explants is also inhibited by serotonin and carbachol. The serotonin effect appears to be mediated by 5-HT1A receptors, based on relative agonist and antagonist selectivity. Chronic exposure of cultures to morphine results in enhanced basal and forskolin-stimulated AC as well as attenuation of opioid-inhibition of AC assayed in the presence of forskolin; treatment of explants with pertussis toxin causes similar changes in the AC system. The inhibitory effect of serotonin is also attenuated by the pertussis toxin treatment. Basal AC activity of the explants (assayed without forskolin present) is stimulated to a small but significant extent by opioids and by serotonin. The opioid stimulatory effect is markedly enhanced following either morphine or pertussis toxin treatment of the explants. The attenuation of opioid- and serotonin-inhibition of AC produced by chronic exposure to pertussis toxin and the attenuation of opioid inhibition produced by exposure to morphine are consonant with the attenuation of opioid and monoaminergic depression of sensory evoked dorsal horn network responses after similar chronic treatments. It is proposed that the inhibitory effects of opioids and serotonin on these neurons are mediated by receptors that are negatively coupled via a pertussis toxin sensitive Gi protein to AC. Furthermore, alterations of AC with chronic morphine treatment may be involved in the development of physiologic tolerance to opioids.

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.

Evidence for loss of brain [3H]spiroperidil and [3H]ADTN binding sites in rabbit brain with aging.

[3H]Spiroperidol and [3H]2-amino-6,7-dihydroxyl-1,2,3,4,-tetrahydronaphthalene hydrochloride (ADTN) binding were measured in various central nervous system regions of 5 month and 5.5 year old rabbits. In striatum, young animals had a 38% higher number of [3H]spiroperidol binding sites and a 140% higher number of [3H]ADTN binding sites than did the older animals. In frontal cortex and anterior limbic cortex there were respectively 42% and 26% more [3H]spiroperidol binding sites in the young animals. There was no change in the binding site number or affinity for [3H]spiroperidol in retina with aging. Pharmacological characterization demonstrated that [3H]spiroperidol binds to a dopamine receptor in striatum and to a serotonin receptor in cortex.

Properties of dopamine agonist and antagonist binding sites in mammalian retina.

Retinal homogenates of calf, rat, rabbit and Cebus appella and Macaca mulata monkeys were found to contain stereospecific binding sites for the dopamine antagonist [3H]spiroperidol. In further studies with calf and rat retina, stereospecific binding sites were also found for the dopamine agonist [3H]ADTN (2-amino-6,7,-dihydroxy-1,2,3,4-tetrahydronapththalene). The [3H]spiroperidol binding sites in calf retina were pharmacologically similar to the dopaminergic spiroperidol binding sites previously demonstrated to be present in striatum. However, calf and rabbit retina contained less than 1/10 the concentration of [3H]spiroperidol binding sites found in striatum. Saturation studies and Scatchard analyses showed a single class [3H]spiroperidol binding sites with Kd (apparent dissociation constant) = 0.3 and 0.2 nM and Bmax (binding site number) = 38 and 24 fmol/mg protein in calf retina and rabbit retina respectively. Rates of [3H]spiroperidol association and dissociation were also evaluated in calf retina. Drug specificity for [3H]ADTN binding in calf retina resembled that previously reported for striatal [3H]ADTN binding and thus differed from retinal [3H]spiroperidol binding. Calf retinal [3H]ADTN binding sites had a Kd = 9 nM and Bmax = 113 +/- 12 fmol/mg protein. Thus, the total number of [3H]ADTN sites in retina was at least twice that of [3H]spiroperidol sites. Guanine nucleotides (GTP and Gpp (NH)p) but not ATP reduced the affinity of the dopamine agonist ADTN for [3H]spiroperidol binding, and also reduced the specific binding of [3H]ADTN itself up to a maximal value of about 50% of control binding. Saturation studies of calf retinal [3H]ADTN binding confirmed that Gpp(NH)p-displaceable sites were a discrete saturable subset of stereospecific [3H]ADTN sites with Kd = 9 nM and Bmax = 50 +/- 6 fmol/mg protein. The Gpp(NH)p insensitive sites had a Kd = 9 nM and Bmax = 63 +/- 7 fmol/mg protein. It is proposed that although [3H]ADTN sites differ pharmacologically from [3H]spiroperidol sites, since [3H]spiroperidol sites are guanine nucleotide-sensitive and similar in number to the guanine nucleotide-sensitive class of [3H]ADTN sites, they may possibly be related to these sites as well as to adenylate cyclase. In addition, retina contains guanine nucleotide-insenstive [3H]ADTN sites, possibly presynaptic and probably not coupled to adenylate cyclase.

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)

Differential affinities of molindone, metoclopramide and domperidone for classes of [3H]spiroperidol binding sites in rat striatum: evidence for pharmacologically distinct classes of receptors.

Rat striatum contains two populations of dopaminergic [3H]spiroperidol binding sites. The two populations are similar in their affinities for chlorpromazine and dopamine. Only one population, that with a somewhat higher affinity for spiroperidol itself, exhibits high affinity for the selective D2 antagonists molindone, metoclopramide and domperidone. Hence, this population may represent D2 receptor sites. The other larger population may represent either a separate class of receptor sites or a different form of D2 receptor sites.

Evidence for selective loss of brain dopamine- and histamine-stimulated adenylate cyclase activities in rabbits with aging.

Dopamine-stimulated adenylate cyclase activity in striatum and both dopamine- and histamine-stimulated adenylate cyclase activity in hypothalamus, frontal cortex and anterior limbic cortex declined by about 50% as rabbits aged from 5.5 months to 5.5 years of age. These changes were primarily in maximal response to amine although an additional component involving decreased affinity in the case of dopamine may also be present. In contrast, dopamine-stimulated adenylate cyclase of retina and both basal and guanyl-5'-yl-imidodiphosphate (Gpp(NH)p)-stimulated activity in these regions were not altered with age. There was no measurable decrease in the old animals in either dopamine or norepinephrine concentration in striatum, anterior limbic cortex or retina, or in choline acetylase activity or [3H]quinuclidinylbenzilate binding in striatum, anterior limbic cortex or frontal cortex. It is proposed that selective age-dependent decreases in transmitter receptors coupled to adenylate cyclases occur in the absence of or independent from neuronal cell loss, as evidenced by the retention of the other biochemical markers.

Catecholamine-stimulated cyclic AMP formation in phenylethanolamine N-methyltransferase containing brain stem nuclei of normal rats and of rats with spontaneous genetic hypertension.

Stimultaion of cyclic AMP formation by epinephrine and norepinephrine has been studied in discrete areas of rat brain that include the epinephrine-containing brain stem nuclei C-1 and C-2. In the C-1 area, epinephrine-stimulated cyclic AMP formation was partially reversed by 100 microM phentolamine and by 10--100 microM propranolol or alprenolol and hence appeared to involve activation of a mixture of both alpha- and beta-adrenergic receptors as has been reported for other rat brain areas such as the cerebral cortex. However, in the C-2-area, the epinephrine and norepinephrine stimulated cyclic AMP formation involved the activation of a single receptor type which was alpha-like in character. Stimulation of cyclic AMP formation by epinephrine in the C-2 area was antagonized by nanomolar concentrations of both phentolamine and yohimbine. The epinephrine-stimulated formation of cyclic AMP in the C-2 but not in the C-1 area was augmented in a strains of rats which exhibit spontaneous genetic hypertension (SHR) vs. Wistar-Kyoto controls. It is suggested that the enhanced epinephrine-stimulated cyclic AMP formation in the C-2 area of SHR rats could be a physiological compensatory response to some other hypertension-causing lesion which, for example, results in chronically reduced epinephrine release or in ruduced availability of epinephrine at its postsynaptic receptor thereby leading to receptor supersensitivity. Supporting this possibility was the finding that treatment of SHRs and control animals and reserpine resulted in enhancement of epinephrine-stimulated cyclic AMP formation in the C-2 area of control rats, essentially obliterating the difference between control and SHR. The findings are also interepreted as supporting the involvement of epinephrine neurons in central vaso-depressor mechanisms.

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.

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).

Retina contains guanine nucleotide sensitive and insensitive classes of dopamine receptors.

Calf retina contains high affinity, stereospecific and saturable binding sites, characterized as dopaminergic, for 3H-spiropheridol (Bmax from Scatchard analysis = 38 +/- 4 fmoles/mg protein) and for 3H-ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene). The 3H-ADTN sites include a discrete and saturable sub-set of sites sensitive to guanine nucleotide inhibition (Bmax = 50 +/- 6 fmoles/mg protein) and a separate saturable sub-set of sites insensitive to guanine nucleotides (Bmax = 63 +/- 7 fmoles/mg protein). The binding of agonists to 3H-spiroperidol sites is also inhibited by guanine nucleotides.

Biogenic amine-stimulated adenylate cyclase and spiroperidol-binding sites in rabbit brain: evidence for selective loss of receptors with aging.

Evidence for selective decreases in biogenic amine receptor function with age in the rabbit has been obtained. Dopamine-stimulated adenylate cyclase activity in the striatum (caudate-putamen) of rabbit brain declined by about 50 percent as rabbits aged from less than 1 to 4 to 5 years of age. Similar decreases in transmitter-stimulated adenylate cyclase activity were found for histamine as well as for dopamine and norepinephrine in hypothalamus, frontal cortex and anterior limbic cortex. Isoproterenol-stimulated activity was also decreased with age in frontal cortex. These changes appeared to represent decreases in maximal response and not alteration in affinity for amine. In contrast, dopamine-stimulated adenylate cyclase of retina and transmitter-independent (basal or Gpp(NH)p-stimulated) activity in each of the regions studied were not altered with age. Dopamine receptors in striatum directly assessed by measurement of [3H]-spiroperidol binding revealed a comparable decrease in the number of binding sites without change in ligand affinity. Preliminary data also indicated decreased spiroperidol binding sites in the cortical regions of older animals. These changes in striatum and cortex were evident in the absence of decreases in either dopamine content or choline acetylase activity, an activity presumed to be present in neurons containing dopamine receptors. It is proposed that selective age-dependent decreases in postsynaptic biogenic amine receptor content occur in the absence of, or independent from, neuronal cell loss, possibly by a mechanism involving receptor desensitization. These changes occur in the animal model in those brain regions which in man are thought to be of importance in the loss of cerebral function that is found with senscence.