Anatomical distribution of mu, delta, and kappa opioid- and nociceptin/orphanin FQ-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate binding in guinea pig brain.

An in vitro autoradiographic technique has recently been developed to visualize receptor-activated G-proteins by using agonist-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding in the presence of excess guanosine 5'-diphosphate. This technique was used to localize opioid-activated G-proteins in guinea pig brain, a species that contains the three major types of opioid receptors. This study used selective mu, delta, and kappa opioid agonists as well as nociceptin or orphanin FQ (N/OFQ) peptide, an endogenous ligand for an orphan opioid receptor-like (ORL1) receptor, to stimulate [35S]GTPgammaS binding in guinea pig brain sections. Opioid receptor specificity was confirmed by blocking agonist-stimulated [35S] GTPgammaS binding with the appropriate antagonists. In general, the distribution of agonist-stimulated [35S]GTPgammaS binding correlated with previous reports of receptor binding autoradiography, although quantitative differences suggest regional variations in receptor coupling efficiency. Mu, delta, and kappa opioid-stimulated [35S]GTPgammaS binding was found in the caudate-putamen, nucleus accumbens, amygdala, and hypothalamus. Mu-stimulated [35S]GTPgammaS binding predominated in the hypothalamus, amygdala, and brainstem, whereas kappa-stimulated [35S]GTPgammaS binding was particularly high in the substantia nigra and cortex and was moderate in the cerebellum. N/OFQ-stimulated [35S] GTPgammaS binding was highest in the cortex, hippocampus, and hypothalamus and exhibited a unique anatomical distribution compared with opioid-stimulated [35S]GTPgammaS binding. The present study extends previous reports on opioid and ORL1 receptor localization by anatomically demonstrating functional activity produced by mu, delta, and kappa opioid and ORL1 receptor activation of G-proteins.

Lack of evidence of kappa2-selective activation of G-proteins: kappa opioid receptor stimulation of [35S] GTPgammaS binding in guinea pig brain.

Although only one gene for kappa opioid receptors has been cloned to date, kappa1 and kappa2 receptors have been defined pharmacologically, with drugs such as bremazocine binding to both putative kappa receptor subtypes. To examine whether kappa receptor subtypes can be distinguished at the level of the G-protein, the ability of the kappa1 agonist (trans-(dl)-3,4-dichloro-N- methyl-N-[2-(1 -pyrrolidinyl)cyclohexyl]-benzeneacetamide) methane sulfonate (U-50488H) to stimulate [35S]guanosine-5'-O-(gamma-thio)-triphosphate ([35S]GTPgammaS) binding in guinea pig brain was compared with that of bremazocine and dynorphin. In membranes prepared from guinea pig striatum, both bremazocine and U-50488H stimulated [35S]GTPgammaS binding with the same relative efficacy, while dynorphin produced at least two-fold greater efficacy than the other two agonists. In vitro autoradiography of agonist-stimulated [35S]GTPgammaS binding revealed similar regional distributions of bremazocine- and U-50488H-activated G-proteins. In striatal membranes, the kappa antagonist nor-binaltorphimine (nor-BNI) blocked both bremazocine- and U-50488H-stimulated [35S]GTPgammaS binding with similar Ke values. In agonist additivity experiments, the stimulation of [35S]GTPgammaS binding by the delta agonist [D-pen2'5, p-Cl-Phe4]enkephalin (p-Cl-DPDPE) was approximately additive with the two kappa agonists. Stimulation of [35S]GTPgammaS binding by the mu agonist [D-Ala2, N-Me4, Gly5-ol]-enkephalin (DAMGO) was additive with U-50488H, but not with bremazocine, reflecting the mu antagonist properties of this compound. The combination of bremazocine and U-50488H together produced no greater stimulation of binding than either agonist alone, indicating that they were binding to the same site. These results demonstrate that bremazocine and U-50488H activate G-proteins in guinea pig brain through the same receptor, and suggest that kappa2 receptors are not coupled through the same signal transduction mechanisms as kappa1 receptors.

Effects of long-term exposure to delta9-THC on expression of cannabinoid receptor (CB1) mRNA in different rat brain regions.

The time course of changes across 21 days of continuous exposure to Delta9-tetrahydrocannabinol (Delta9-THC) was assessed for the level of cannabinoid receptor (CB1) mRNA expression in three different rat brain regions: cerebellum, hippocampus and corpus striatum. Expression levels of CB1 mRNA were determined using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) following a protocol which included a gene standard, 28S ribonucleic acid protein (rRNA), for normalization of levels of RNA in the three different brain regions. The levels of CB1 mRNA were assessed in four different rats at each of seven time points (6 h, and 1, 2, 3, 7, 14 and 21 days) during a 21-day Delta9-THC one dose day-1 (10 mg kg-1) treatment regimen. In the cerebellum and hippocampus, CB1 mRNA levels were increased above vehicle control animals at 7 and 14 days of treatment. In the striatum the levels of CB1 transcripts were severely reduced from days 2-14. CB1 message expression in all three brain areas returned to vehicle control levels by day 21 of Delta9-THC treatment, a time at which behavioral tolerance has been previously reported. An additional measure, receptor stimulated GTPgammaS binding, performed over the same time period revealed differential desensitization within the 3 brain areas as a function of chronic exposure to Delta9-THC. Hippocampus was the earliest to desensitize decreasing to 35% of control by treatment day 7, followed by a decrease in the cerebellum to that same level on day 14 of treatment. The striatum showed only half that degree of desensitization (65%) over the entire 21-day treatment period. Comparisons suggests that CB1 message may be regulated by different effector systems in each of the three areas during chronic Delta9-THC exposure.

Arcuate nucleus projections to brainstem regions which modulate nociception.

Anterograde tracing studies were conducted in order to identify efferents from the arcuate nucleus, which contains the hypothalamic opiocortin neuronal pool. Phaseolus vulgaris leucoagglutinin (PHA-L) was stereotaxically iontophoresed into the arcuate nucleus and the terminal fields emanating from the labelled perikarya were identified immunocytochemically. PHA-L-immunoreactive (-ir) fibers were identified in nucleus accumbens, lateral septal nucleus, bed nucleus of the stria terminalis, medial and lateral preoptic areas, anterior hypothalamus, amygdaloid complex, lateral hypothalamus, paraventricular nucleus, zona incerta, dorsal hypothalamus, periventricular gray, medial thalamus and medial habenula. In the brainstem, arcuate terminals were identified in the periaqueductal gray (PAG), dorsal raphe nucleus (DRN), nucleus raphe magnus (NRM), nucleus raphe pallidus, locus coeruleus, parabrachial nucleus, nucleus reticularis gigantocellularis pars alpha, nucleus tractus solitarius and dorsal motor nucleus of the vagus nerve. Dual immunostaining was used to identify the neurochemical content of neurons in arcuate terminal fields in the brainstem. Arcuate fiber terminals established putative contacts with serotonergic neurons in the ventrolateral PAG, DRN and NRM and with noradrenergic neurons in periventricular gray, PAG and locus coeruleus. In the PAG, arcuate fibers terminated in areas with neurons immunoreactive to substance P, neurotensin, enkephalin and cholecystokinin (CCK) and putative contacts were identified with CCK-ir cells. This study provides neuroanatomical evidence that putative opiocortin neurons in the arcuate nucleus influence a descending system which modulates nociception.

Identification of opioid receptor-like (ORL1) peptide-stimulated [35S]GTP gamma S binding in rat brain.

Recent reports have identified an endogenous peptide ligand for the opioid receptor-like (ORL1) receptor. In the present study, ORL1 peptide-stimulated [35>]GTP gamma S binding was assessed in rat cortical membranes and brain sections to localize ORL1 receptor-activated G-proteins. In membrane assays, with 20 microM GDP, ORL1 peptide stimulated [35S]GTP gamma S binding by approximately two-fold with an ED50 value of 20 nM. ORL1 peptide-stimulated [35S]GTP gamma S binding was unaffected by opioid or other G-protein-coupled receptor antagonists. In brain sections, ORL1 peptide-stimulated [35S]GTP gamma S binding was identified in regions including cortex, amygdala, hypothalamus, thalamus and brain stem. The anatomical distribution of ORL1 peptide-stimulated [35S]GTP gamma S binding suggests its involvement in cognition, emotion and homeostasis.

Dorsal raphe nucleus efferents: termination in peptidergic fields.

This study was performed to identify the efferents of the dorsal raphe nucleus (DRN), particularly in regard to nociception. Phaseolus vulgaris leucoagglutinin (PHA-L) was microiontophoresed into the DRN and visualized immunocytochemically; PHA-L-immunoreactive (-IR) fibers and terminals were identified in the forebrain, hypothalamus, midline and intralaminar thalamus, habenula, periaqueductal gray, locus coeruleus, parabrachial nucleus, medullary raphe and reticular nuclei, and nucleus tractus solitarius. Dual immunocytochemistry was used to identify corticotropin-releasing factor, neurotensin, and enkephalin neurons in DRN terminal fields, in some cases in close proximity to PHA-L-IR terminals. Terminal fields were identified in regions that influence nociception, and the neuroactive substances identified in these terminal fields may play modulatory roles in nociception.

Efferents of the opiocortin-containing region of the commissural nucleus tractus solitarius.

Efferent projections of the commissural nucleus tractus solitarius (cNTS0 in the region containing opiocortin-immunoreactive (-IR) neurons were identified using Phaseolus vulgaris leucoagglutinin (PHA-L). Efferents were identified in the bed nucleus of the stria terminalis, preoptic area, amygdala, hypothalamus, periaqueductal gray, parabrachial nucleus, locus coeruleus, medullary catecholaminergic groups, and NTS. The PHA-L-IR varicosities in lateral parabrachial nucleus were identified in close association with CRF-IR and enkephalin-IR cells. These data on cNTS projections are consistent with our previous immunocytochemical and lesion studies on opiocortin connectivity and provide anatomical evidence that neurons in the cNTS may influence cardiovascular and sympathetic nervous system function via connectivity with nuclei in the lateral brain stem.

Serotonin and substance P afferents to parafascicular and central medial nuclei.

Potent analgesia is elicited by electrical stimulation of the periaqueductal gray (PAG), dorsal raphe nucleus (DRN) and intralaminar thalamus. Horseradish peroxidase conjugated wheat germ agglutinin (HRP-WGA) was stereotaxically pressure injected into the parafascicular (PF) or central medial (CM) nucleus to identify brainstem afferents to the intralaminar thalamus. WGA-immunoreactive (-ir) neurons were identified in the DRN, PAG and lateral dorsal tegmentum (LDTg) after PF and CM injections. Many retrogradely labeled cells in the DRN and ventral PAG were also serotonin-ir, and a portion of WGA-ir cells in the LDTg were substance P-ir. These results substantiate previous studies implicating the intralaminar thalamus and periaqueductal region, as well as serotonin and substance P, in antinociception.

Opiocortin and catecholamine projections to raphe nuclei.

Afferent projections to the nucleus raphe magnus (NRM) and dorsal raphe nucleus (DRN) were identified using retrograde transport of horseradish peroxidase conjugated wheat germ agglutinin (HRP-WGA). Neurons were labeled in important nociceptive regions including periaqueductal gray (PAG), arcuate nucleus, lateral hypothalamus and medial thalamic nuclei following both injections. We have immunocytochemically identified opiocortin/WGA neurons in the arcuate nucleus following NRM and DRN injections. Dual stained catecholamine/WGA perikarya were found in zona incerta, locus coeruleus, substantia nigra, nucleus tractus solitarius and adjacent A2, C2 and C3, lateral paragigantocellular reticular nucleus/C1 and lateral reticular nucleus/A1 following DRN injections and in zona incerta, substantia nigra, nucleus tractus solitarius/A2 and lateral reticular nucleus/A1 after NRM injections. These results provide further evidence for opiocortin and catecholamine modulation of analgesia.

Calcium and cAMP mediated stimulation of Fos in cultured hypothalamic tyrosine hydroxylase-immunoreactive neurons.

Immediate-early genes, such as c-fos, couple extracellular signals to genetic changes in the cell. We have previously demonstrated that depolarization with 50 mM KCl increases Fos immunoreactivity in hypothalamic tyrosine hydroxylase (TH) and oxytocin immunoreactive (-ir) neurons in primary culture. This Fos activation occurs within 1.5-2 h in TH-ir cells. In the present study, we examined the effects of depolarization, glutamate receptor activation and adenylyl cyclase stimulation on Fos-ir to determine the possible mechanism(s) of Fos activation in TH-ir neurons. Hypothalamic cultures were treated with KCl, glutamate or forskolin, and Fos and TH were visualized immunocytochemically. Forskolin increased the percentage of Fos/TH-ir neurons in a dose-dependent manner, with a maximal stimulation of 53.4 +/- 4.5% Fos/TH-ir neurons at 30 microM forskolin. The dose-response curve for glutamate was steep, with a maximal stimulation of 24.8 +/- 2.1% Fos-ir neurons at 100 microM. 50 mM KCl resulted in 50.0 +/- 0.8% Fos/TH-ir neurons. Pretreatment with verapamil decreased KCl induced Fos-ir by 57%, glutamate by 65% and forskolin by 39%. Combined drug administration demonstrated significant additivity between forskolin and glutamate, and forskolin and KCl, however, no significant additivity was found with KCl and glutamate. The results are discussed in terms of cAMP and calcium mediation of the Fos response to these stimuli.

D2 inhibition of stimulated Fos immunoreactivity in cultured tyrosine hydroxylase-ir hypothalamic neurons.

We have previously demonstrated that Fos immunoreactivity can be stimulated by KCl, forskolin or glutamate in cultured tyrosine hydroxylase-immunoreactive (TH-ir) hypothalamic neurons. The present study was performed to determine whether agents that regulate dopaminergic activity, particularly D1 and D2 receptor agonists, modulate the intracellular cascade leading to Fos expression. Dissociated hypothalamic cultures were prepared from neonatal rats. The cultures were treated with D1- or D2-specific agonists, followed by KCl, forskolin or glutamate. Cultures were fixed after 2 h and immunocytochemically stained for tyrosine hydroxylase and Fos. Pretreatment of the cultures with the D2 agonist LY163502 inhibited KCl- and forskolin-stimulated Fos-ir in TH-ir neurons in a saturable dose-dependent manner. The maximal effective dose was 30 microM LY163502, which decreased Fos-ir by 23% in cultures treated with 50 mM KCl and by 33% in those treated with 30 microM forskolin. The D2 agonist had no effect on glutamate-stimulated Fos-ir. LY163502 inhibition of Fos-ir was blocked by D2 antagonist or Bordetella pertussis toxin pretreatment which demonstrates that the effect is mediated by D2 receptor activation of an inhibitory G protein. Treatment of the cultures with the D1 agonist SKF82526 had no effect on basal or stimulated levels of Fos-ir. These results demonstrate that in neonatal TH-ir hypothalamic neurons the D2 receptor system may regulate levels of the immediate-early gene product Fos and, therefore, subsequent genetic expression in these neurons.

Differences in G-protein activation by mu- and delta-opioid, and cannabinoid, receptors in rat striatum.

Receptor activation of G-proteins can be measured by agonist-stimulated [35S]GTP gamma S binding in the presence of excess guanosine diphosphate (GDP). To determine whether opioid and cannabinoid receptor-mediated G-protein activation correlate with their receptor densities, this study compared opioid- and cannabinoid-stimulated [35S]guanylyl-5'-O-(gamma-thio)-triphosphate (GTP gamma S) binding with the corresponding Bmax values of receptor binding in rat striatum. Scatchard analysis revealed that the Bmax of cannabinoid receptor binding was approximately ten times higher than that of mu- or delta-opioid receptor binding. However, comparable levels of cannabinoid- and mu- and delta-opioid-stimulated [35S]GTP gamma S binding were observed in the caudate-putamen by [35S]GTP gamma S autoradiography in brain sections. Scatchard analysis of net agonist-stimulated [35S]GTP gamma S binding in membranes showed that the Bmax of cannabinoid-stimulated binding was only twice that of mu- or delta-opioid-stimulated binding. Thus, the calculated amplification factors for mu- and delta-opioid receptors are seven times that of cannabinoid receptors.

Fos activation in cultured tyrosine hydroxylase and oxytocin immunoreactive neurons.

Fos and other immediate early gene products are used as markers for neuronal activity. We identified Fos immunocytochemically after KCI-induced depolarization of cultured hypothalamic neurons. Five-day cultures were treated for 1 h with 50 mM KCI or media and fixed at 0, 0.5, 1, 2, and 4 h posttreatment. Sequential immunocytochemistry was performed to identify Fos immunoreactivity in tyrosine hydroxylase (TH)-immunoreactive (-ir) or oxytocin (OT)-ir neurons. Activated neurons [brown cells (TH-ir or OT-ir) with purple nuclei (Fos-ir)] were counted microscopically. KCI treatment resulted in an increased percentage of Fos-ir TH-ir and OT-ir neurons over control levels over the time course examined. Fos-ir peaked in TH-ir neurons at 0.5 to 1 h posttreatment, with levels returning to baseline at 4 h. Fos-ir in OT-ir neurons peaked at 2 h, and remained elevated at 4 h, showing prolonged activation. These results demonstrate that KCI-induced depolarization of cultured hypothalamic neurons increases Fos with a different time course in TH-ir vs. OT-ir neurons.

Efferent projections of the nucleus raphe magnus.

This study was performed to identify supraspinal efferents of the nucleus raphe magnus (NRM) in the rat using the anterograde tracer phaseolus vulgaris leucoagglutinin (PHA-L). NRM-derived PHA-L-ir fibers, with putative terminals, were identified in regions including the lateral hypothalamus, parafascicular nucleus, ventral lateral periaqueductal gray (PAG), locus coeruleus, parabrachial nucleus, A7, A5, and nucleus tractus solitarius. Projections to the PAG demonstrate reciprocity in PAG-NRM connectivity that may modulate the PAG-NRM-spinal cord pathway. The NRM may contribute to supraspinal modulation of nociception by efferents identified in the PAG, as well as locus coeruleus, A7, and A5, which have been shown to project to the spinal cord dorsal horn. Our results provide neuroanatomical support for NRM involvement in supraspinal mechanism(s) for modulation of nociception.

In vitro autoradiographic localization of 5-HT1A receptor-activated G-proteins in the rat brain.

Serotonin 5-HT1A receptors belong to the superfamily of G-protein-coupled receptors. Receptor activation of G-proteins can be determined by agonist-stimulated [35S]GTPgammaS binding in the presence of excess GDP, and in vitro autoradiographic adaptation of this technique allows visualization of receptor-activated G-proteins in tissue sections. The present study was performed to examine 5-HT1A receptor activation of G-proteins using 8-OH-DPAT-stimulated [35S]GTPgammaS binding in membranes and brain sections. In hippocampal membranes, 8-OH-DPAT stimulated [35S]GTPgammaS binding by twofold, with an ED50 value of 25 nM. 5-HT1 antagonists, but not 5-HT2 antagonists, increased the ED50 of 8-OH-DPAT in a manner consistent with competitive antagonists. Scatchard analysis of [35S]GTPgammaS binding showed that 8-OH-DPAT induced the formation of high affinity [35S]GTPgammaS binding sites with a KD for GTPgammaS of 3.2 nM. [35S]GTPgammaS autoradiography, performed in brain sections with the 5-HT1A agonist 8-OH-DPAT, revealed high levels of 5-HT1A-stimulated [35S]GTPgammaS binding in the hippocampus, lateral septum, prelimbic cortex, entorhinal cortex, and dorsal raphe nucleus. 5-HT1A-stimulated [35S]GTPgammaS binding in sections was blocked by the addition of the 5-HT1 antagonist methiothepin. These results show that the use of agonist-stimulated [35S]GTPgammaS autoradiography for the 5-HT1A receptor system should provide new information regarding signal transduction in specific brain regions.

Cannabinoid receptor stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding in rat brain membranes.

Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTP gamma S) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [35S]GTP gamma S binding in rat brain membranes. Assays were conducted with 0.05 nM [35S]GTP gamma S, incubated with rat cerebellar membranes, 1-30 microM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [35S]GTP gamma S binding increased with increasing concentrations of GDP, with 10-30 microM GDP providing approximately 150-200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [35S]GTP gamma S binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [35S]GTP gamma S binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [35S]GTP gamma S binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [35S]GTP gamma S autoradiography. These results demonstrate that [35S]GTP gamma S binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.

In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5'-[gamma-[35S]thio]-triphosphate binding.

Agonists stimulate guanylyl 5'-[gamma-[35S]thio]-triphosphate (GTP[gamma-35S]) binding to receptor-coupled guanine nucleotide binding protein (G proteins) in cell membranes as revealed in the presence of excess GDP. We now report that this reaction can be used to neuroanatomically localize receptor-activated G proteins in brain sections by in vitro autoradiography of GTP[gamma-35S] binding. Using the mu opioid-selective peptide [D-Ala2,N-MePhe4,Gly5-ol]enkephalin (DAMGO) as an agonist in rat brain sections and isolated thalamic membranes, agonist stimulation of GTP[gamma-35S] binding required the presence of excess GDP (1-2 mM GDP in sections vs. 10-30 microM GDP in membranes) to decrease basal G-protein activity and reveal agonist-stimulated GTP[gamma-35S] binding. Similar concentrations of DAMGO were required to stimulate GTP[gamma-35S] binding in sections and membranes. To demonstrate the general applicability of the technique, agonist-stimulated GTP[gamma-35S] binding in tissue sections was assessed with agonists for the mu opioid (DAMGO), cannabinoid (WIN 55212-2), and gamma-aminobutyric acid type B (baclofen) receptors. For opioid and cannabinoid receptors, agonist stimulation of GTP[gamma-35S] binding was blocked by incubation with agonists in the presence of the appropriate antagonists (naloxone for mu opioid and SR-141716A for cannabinoid), thus demonstrating that the effect was specifically receptor mediated. The anatomical distribution of agonist-stimulated GTP[gamma-35S] binding qualitatively paralleled receptor distribution as determined by receptor binding autoradiography. However, quantitative differences suggest that variations in coupling efficiency may exist between different receptors in various brain regions. This technique provides a method of functional neuroanatomy that identifies changes in the activation of G proteins by specific receptors.

Regional differences in cannabinoid receptor/G-protein coupling in rat brain.

Cannabinoid receptor activation of G-proteins can be measured by WIN 55212-2-stimulated [35S]GTPgammaS binding. Receptor/transducer amplification factors, interpreted as the number of G-proteins activated per occupied receptor, are the ratio of the apparent B(max) of net agonist-stimulated [35S]GTPgammaS binding to the B(max) of receptor binding. The present study examined whether amplification factors for cannabinoid receptors differ among various rat brain regions. In autoradiographic studies with [3H]WIN 55212-2 and WIN 55212-2-stimulated [35S]GTPgammaS binding, some regions displayed different relative levels of agonist-stimulated [35S]GTPgammaS binding than receptor binding. To quantify amplification factors, membranes from different brain regions were assayed by saturation binding analysis of net WIN 55212-2-stimulated [35S]GTPgammaS, [3H]SR141716A (antagonist) and [3H]WIN 55212-2 (agonist) binding. For [3H]SR141716A binding, amplification factors varied significantly from 2.0 (frontal cortex) to 7.5 (hypothalamus). For [3H]WIN 55212-2 binding, amplification factors ranged from 2.4 (hippocampus) to 5.5 (thalamus). Comparison of receptor binding and G-protein activation at subsaturating concentrations of WIN 55212-2 indicated that amplification factors may vary with receptor occupancy in some regions like cerebellum. Ratios between high-affinity [3H]WIN 55212-2 B(max) and [3H]SR141716A B(max) also differed significantly among brain regions. These results demonstrate that G-protein coupling by cannabinoid receptors differs among brain regions, and therefore depends on the cellular environment.

Dissociation between vasopressin and oxytocin mRNA and peptide secretion after AV3V lesions.

The effect of hypertonic NaCl consumption on vasopressin (VP) and oxytocin (OT) mRNA levels and plasma and pituitary peptides was evaluated in rats with sham or anterior ventral third ventricular (AV3V) lesions. Rats were given tap water or 2% NaCl for 4 days. Because the rats with lesions drank significantly less salt solution than the controls (78.8 +/- 17.4 vs. 205.5 +/- 37.8 ml/4 days), a second control group was included in which saline intake was matched to the lesioned group. AV3V rats showed a deficit in the peptide response to the osmotic stimulus. There was no increase in plasma VP or OT or decrease in posterior pituitary peptide content in the face of an extreme hypernatremia: plasma sodium of 180.1 +/- 4.2 meq/l. Evaluation of mRNA changes by means of in situ hybridization showed that animals with lesions responded to the salt challenge with increases in hypothalamic VP and OT mRNA levels. There were significant increases in paraventricular and supraoptic OT mRNA and paraventricular VP mRNA in the lesioned group. The salt-matched control group showed no changes in peptide mRNA levels. These results demonstrate that AV3V lesions produce an impairment of the salt-neuroendocrine reflex but a persistence of the peptide mRNA response. Differences in control mechanisms must account for this dissociation between peptide mRNA expression and peptide secretion.