Novel time-dependent vascular actions of Delta9-tetrahydrocannabinol mediated by peroxisome proliferator-activated receptor gamma

Cannabinoids have widespread effects on the cardiovascular system, only some of which are mediated via G-protein-coupled cell surface receptors. The active ingredient of cannabis, Delta9-tetrahydrocannabinol (THC), causes acute vasorelaxation in various arteries. Here we show for the first time that THC also causes slowly developing vasorelaxation through activation of peroxisome proliferator-activated receptors gamma (PPARgamma). In vitro, THC (10 microM) caused time-dependent vasorelaxation of rat isolated arteries. Time-dependent vasorelaxation to THC was similar to that produced by the PPARgamma agonist rosiglitazone and was inhibited by the PPARgamma antagonist GW9662 (1 microM), but not the cannabinoid CB1 receptor antagonist AM251 (1 microM). Time-dependent vasorelaxation to THC requires an intact endothelium, nitric oxide, production of hydrogen peroxide, and de novo protein synthesis. In transactivation assays in cultured HEK293 cells, THC-activated PPARgamma, transiently expressed in combination with retinoid X receptor alpha and a luciferase reporter gene, in a concentration-dependent manner (100 nM-10 microM). In vitro incubation with THC (1 or 10 microM, 8 days) stimulated adipocyte differentiation in cultured 3T3L1 cells, a well-accepted property of PPARgamma ligands. The present results provide strong evidence that THC is a PPARgamma ligand, stimulation of which causes time-dependent vasorelaxation, implying some of the pleiotropic effects of cannabis may be mediated by nuclear receptors.     

Evidence that the stimulus properties of apomorphine are mediated by both D1 and D2 receptor activation

Male and female rats were trained to discriminate between the stimulus properties of apomorphine (0.16 mg/kg i.p.) and saline in a two-lever, food-motivated operant procedure. Apomorphine, at doses different than the training dose, produced a similar dose-dependent relationship in both sexes. Consistent with the hypothesis that the behavioral effects of apomorphine are mediated by D2 activation, the apomorphine interoceptive cue generalized to bromocriptine, a drug considered to be a preferential D2 agonist. In addition, the dose-response curve after 5-15 mg/kg bromocriptine administration was parallel to that of apomorphine. Consistent with the biochemical evidence that apomorphine's effects are mediated, to a lesser extent, by D1 activation, the apomorphine cue partially generalized to the selective D1 agonist SKF 38393. Furthermore, the apomorphine cue was not blocked by the selective D1 antagonist SCH 23390. Somewhat surprising was the partial generalization of the apomorphine cue to SCH 23390. However, this is not the first time that the administration of SCH 23390 has resulted in unexpected behavioral responses. Other novel findings include the lack of sex differences in acquisition training to the apomorphine cue and in the generalization tests to the selective agonists. The behavioral results are consistent with previous biochemical evidence that the effects of apomorphine are mediated by both D1 and D2 activation and is further behavioral support that apomorphine's effects are not the result of D2 activation alone, as previously hypothesized.     

Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism

Leu-enkephalin (100 micrograms/kg, i.p.) administered to mice 5 min before training in a one way active avoidance task significantly reduced the number of avoidances observed in the peptide treated animals. This impairing action of Leu-enkephalin was partially attenuated by methylnaloxonium (naloxonium), a quarternary form of naloxone with a limited ability to penetrate the blood brain barrier. Passive immunization (i.v.) of mice with a Leu-enkephalin antiserum 4 hrs before training produced an effect on avoidance conditioning that was the opposite to that observed with Leu-enkephalin alone. That is, passive immunization increased the number of avoidances observed in the treated mice. The results suggest that Leu-enkephalin actions on avoidance conditioning are mediated by a peripheral opioid mechanism, that leu-enkephalin may have a primary site of action outside the blood brain barrier, and that peripheral Leu-enkephalin systems may normally operate to influence conditioned avoidance behavior.     

Insulin action on adipocytes. Evidence that the anti-lipolytic and lipogenic effects of insulin are mediated by the same receptor.

1. The dose-response relationships of insulin stimulation of lipogenesis and inhibition of lipolysis were studied simultaneously by using rat adipocytes to determine whether these different effects of insulin are mediated through the same or different sets of receptors. 2. The sensitivity (defined as the concentration of insulin required to produce a half-maximal effect) of the stimulated lipogenic response to insulin was not significantly different from the sensitivity of the anti-lipolytic response to insulin. The addition of different adrenaline and glucose concentrations did not alter the half-maximal concentration of insulin required to inhibit lipolysis. 3. The specificities of the lipogenic and antilipolytic responses were studied by using insulin analogues. The sensitivities of the lipogenic and anti-lipolytic responses were the same for five chemically modified insulins and hagfish insulin, which have potencies compared with bovine insulin of between 3 and 90%. 4. Starving rats for 48h significantly increased the sensitivities of both the antilipolytic and lipogenic responses to insulin, but the changes in the sensitivities of both lipogenesis and anti-lipolysis returned to that of fed rats. 5. We conclude that insulin stimulates lipogenesis and inhibits lipolysis over the same concentration range. These observations provide powerful evidence that the different effects of insulin are mediated through the same set of receptors.     

Digestive motor effects and vascular actions of CGRP in dog are expressed by different receptor subtypes

Calcitonin gene-related peptide (CGRP) is a 37 AA peptide localized in blood vessels and nerves of the GI tract. Activation of CGRP receptors (subtypes 1 or 2) usually induces vasodilation and/or muscle relaxation, but its effects in dog and on gastroduodenal motility are still unclear. This study looked for the effect of CGRP and the antagonist CGRP8-37, specific for CGRP type 1 receptor, 1) on GI motility (interdigestive and postprandial), and 2) on hemodynamy, in conscious dogs. During the interdigestive period, the infusion of CGRP1-37 (200 pmol/kg/h) or CGRP8-37 (2000 pmol/kg/h) did not modify the duration of the migrating motor complex nor the release nor the motor action of plasma motilin. The gastric emptying of a solid meal (15 g meat/kg) was reduced by the administration of CGRP1-37 (AUC: 2196 +/- 288.6 versus 3618 +/- 288.4 with saline or T12: 78 +/- 7.3 versus 50 +/- 4.3 min; P < 0.01) and this effect was reversed by the antagonist CGRP8-37. CGRP1-37 significantly (P < 0. 01) diminished arterial pressures (118 +/- 1.6/64 +/- 1.4 vs. 125 +/- 1.4/75 +/- 1.2 mmHg with saline) and accelerated the basal cardiac rhythm (110 +/- 1.4 versus 83 +/- 1.6 beats/min). However, CGRP8-37 failed to block the cardiovascular effects of CGRP1-37. In dog, CGRP could influence digestive motility by slowing the gastric emptying of a meal through an action on CGRP-1 receptors. Hemodynamic effects of CGRP were not blocked by CGRP8-37 and seem therefore mediated by CGRP-2 receptor subtype.     

Anorectic actions of prolactin-releasing peptide are mediated by corticotropin-releasing hormone receptors

Prolactin-releasing peptide (PrRP) reduces food intake and body weight and modifies body temperature when administered centrally in rats, suggesting a role in energy homeostasis. However, the mediators of PrRP's actions are unknown. The present study, therefore, first examined the possible involvement of the anorectic neuropeptides corticotropin-releasing hormone (CRH) and the melanocortins (e.g., alpha-melanocyte-stimulating hormone) in PrRP's effects on food intake and core body temperature and, second, determined if PrRP affects energy expenditure by measuring oxygen consumption (Vo2). Intracerebroventricular injection of PrRP (4 nmol) to 24-h-fasted male Sprague-Dawley rats decreased food intake and modified body temperature. Blockade of central CRH receptors by intracerebroventricular coadministration of the CRH receptor antagonist astressin (20 microg) reversed the PrRP-induced reduction in feeding. However, astressin's effect on PrRP-induced changes in body temperature was complicated because the antagonist itself caused a slight rise in body temperature. In contrast, intracerebroventricular coadministration of the melanocortin receptor-3/4 antagonist SHU-9119 (0.1 nmol) had no effect on any of PrRP's actions. Finally, intracerebroventricular injection of PrRP (4 nmol) caused a significantly greater Vo2 over a 3-h test period compared with vehicle-treated rats. These results show that the anorectic actions of PrRP are mediated by central CRH receptors but not by melanocortin receptors-3/4 and that PrRP can modify Vo2.     

Evidence that the effects of arginine-8-vasopressin (AVP) on pituitary corticotropin (ACTH) release are mediated by a novel type of receptor

Ovine corticotropin releasing factor (oCRF-41) and AVP act synergistically to stimulate pituitary ACTH secretion. In the present study we have investigated whether the effect of AVP, either in the presence or in the absence of oCRF-41 (0.5 nmol/l), could be blocked by V1 (pressor)-antagonists. Furthermore, oxytocin, and [1-deamino,8-D-arginine] vasopressin (dDAVP) were tested for their ability to release ACTH. All experiments were carried out in vitro, using segments of rat anterior pituitary glands. The V1-antagonist [1-deamino,penicillamine(o-methyl-tyrosine)]AVP inhibited ACTH release induced by AVP or AVP + oCRF-41. However, it also had some agonistic activity which was more pronounced in the presence of oCRF-41. An equally potent V1-antagonist, [1-beta-mercapto-beta, beta-cyclopentamethyleneproprionic acid (o-methyl-tyrosine)]AVP, failed to inhibit AVP-stimulated ACTH secretion, and also had weak agonist potency. The relatively selective V2 (antidiuretic)-agonist dDAVP was 20-30 fold less potent than AVP. Oxytocin, a weak V1- and V2-agonist was only 4-8 fold less potent than AVP. These data are compatible with the suggestion that AVP receptors on pituitary corticotrope cells are neither classical V1- nor V2-receptors.     

GnRH actions on rat preovulatory follicles are mediated by paracrine EGF-like factors

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG.     

Evidence that v-Src-induced phospholipase D activity is mediated by a G protein.

v-Src-induced increases in diglyceride are derived from phosphatidylcholine via a type D phospholipase (PLD) and a phosphatidic acid phosphatase. v-Src-induced PLD activity, as measured by PLD-catalyzed transphosphatidylation of phosphatidylcholine to phosphatidylethanol, is inhibited by GDP beta S, which inhibits G-protein-mediated intracellular signals. Similarly, v-Src-induced increases in diglyceride are also blocked by GDP beta S. In contrast to the PLD activity induced by v-Src, PLD activity induced by the protein kinase C agonist, 12-O-tetradecanoylphorbol-13-acetate (TPA), was insensitive to GDP beta S. Consistent with the involvement of a G protein in the activation of PLD activity by v-Src, GTP gamma S, a nonhydrolyzable analog of GTP that potentiates G-protein-mediated signals, strongly enhanced PLD activity in v-Src-transformed cells relative to that in parental BALB/c 3T3 cells. The effect of GTP gamma S on PLD activity in v-Src-transformed cells was observed only when cells were prelabeled with [3H]myristate, which is incorporated exclusively into phosphatidylcholine, the substrate for the v-Src-induced PLD. There was no difference in the effect of GTP gamma S-induced PLD activity on v-Src-transformed and BALB/c 3T3 cells when the cells were prelabeled with [3H]arachidonate, which is not incorporated into phospholipids that are substrates for the v-Src-induced PLD. Similarly, GDP beta S inhibited PLD activity in v-Src-transformed cells much more strongly than in BALB/c 3T3 cells when [3H]myristate was used to prelabel the cells. The GTP-dependent activation of PLD by v-Src was dependent upon the presence of ATP but was unaffected by either cholera or pertussis toxin. These data suggest that v-Src induces PLD activity through a phosphorylation event and is mediated by a cholera and pertussis toxin-insensitive G protein.     

Biologic activities of naturally occurring human insulin receptor mutations. Evidence that metabolic effects of insulin can be mediated by a kinase-deficient insulin receptor mutant.

We have studied insulin receptor-mediated signaling in Chinese hamster ovary (CHO) cell transfectants that expressed either of two naturally occurring mutant human insulin receptors: Trp1200----Ser1200 and Ala1134----Thr1134. Compared with overexpressed normal human insulin receptors, both mutant receptors displayed normal processing and normal binding affinity; however, neither was capable of detectable insulin-stimulated autophosphorylation or tyrosine kinase activity toward endogenous (pp185) or exogenous substrates. Several biologic actions of insulin were evaluated in transfected cells. Compared with neomycin-only transfected CHO cells (CHO-NEO), cells expressing normal receptors demonstrated increased insulin sensitivity for 2-deoxyglucose uptake, [14C]glucose incorporation into glycogen, [3H]thymidine incorporation into DNA, and specific gene expression (accumulation of glucose transporter GLUT-1 mRNA). Cells expressing either Ser1200 or Thr1134 receptors showed no increase in insulin-stimulated thymidine incorporation or GLUT-1 mRNA accumulation compared with CHO-NEO. Surprisingly, cells expressing Ser1200 receptors showed increased insulin stimulation of 2-deoxyglucose uptake and glucose incorporation into glycogen compared with CHO-NEO, whereas Thr1134 receptors failed to signal these metabolic responses. We conclude that 1) transfected kinase-deficient insulin receptor mutants derived from insulin-resistant patients have distinct defects in the ability to mediate insulin action in vitro; 2) divergence of insulin signaling pathways may occur at the level of the receptor; and 3) normal activation of the receptor tyrosine kinase by insulin is not necessarily required for signaling of certain important biologic actions.     

Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor

Efforts to understand how plants respond to aluminum have focused on describing the symptoms of toxicity and elucidating mechanisms of tolerance; however, little is known about the signal transduction steps that initiate the plant's response. Here, we image cortical microtubules and quantify plasma-membrane potential in living, root cells of intact Arabidopsis seedlings. We show that aluminum depolymerizes microtubules and depolarizes the membrane, and that these responses are prevented by calcium channel blockade. Calcium influx might involve glutamate receptors, which in animals are ligand-gated cation channels and are present in the Arabidopsis genome. We show that glutamate depolymerizes microtubules and depolarizes the plasma membrane. These responses, and also the inhibition of root elongation, occur within the first few min of treatment, but are evoked more rapidly by glutamate than by aluminum. Microtubule depolymerization and membrane depolarization, induced by either glutamate or aluminum, are blocked by a specific antagonist of ionotropic glutamate receptors, 2-amino-5-phosphonopentanoate; whereas an antagonist of an aluminum-gated anion channel blocks the two responses to aluminum but not to glutamate. For growth, microtubule integrity, and membrane potential, responses to combined glutamate and aluminum were not greater than to glutamate alone. We propose that signaling in response to aluminum is initiated by efflux of a glutamate-like ligand through an anion channel and the binding of this ligand to a glutamate receptor.     

Evidence from a protein-coding gene that acanthocephalans are rotifers

Abstract. Rotifera and Acanthocephala are generally regarded as separate phyla sharing a basal position among triploblast protostomes. This paper presents the first molecular phylogenetic examination of the relationship of Acanthocephala to all three rotifer classes, Seisonidea, Monogononta, and Bdelloidea. Inclusion of Acanthocephala within Rotifera, probably as a sister-taxon to a clade composed of Bdelloidea and Monogononta (the Eurotatoria), is strongly supported by both parsimony and distance methods, using a region of the nuclear coding gene hsp82. Previous molecular evidence for the inclusion of Acanthocephala in the Rotifera suggested that Acanthocephala is a sister-taxon of Bdelloidea, forming the clade Lemniscea. No support is found for this clade, and evidence is presented that the monogonont rotifer used in those analyses, Brachionus plicatilis , may be evolving in an anomalous manner.     

Evidence that natural cytotoxicity and antibody-dependent cellular cytotoxicity are mediated in humans by the same effector cell populations.

The present study strongly suggests that, in humans, natural killer (NK) activity and antibody-dependent cell-mediated cytotoxicity (ADCC) are mediated by the same effector cell population. This is supported by two different experimental approaches. First, competition for NK effector cells was accompanied by simultaneous inhibition of ADCC activity. Target cells sensitive to NK activity were capable of inhibiting specifically an ADCC assay in cold target competition experiments. Second, specific removal of NK cells on monolayers formed by target cells sensitive to NK activity caused simultaneous depletion of ADCC effector cells. In association with the removal on the monolayers of effector cells for ADCC as well as NK activity, we also found a significant depletion of cells bearing Fc gamma receptors.     

Evidence that bears are induced ovulators

The objective of this study was to determine if black bears are induced ovulators. We conducted a single experiment with two replicates; each replicate was divided into two arms: females exposed to male bears and females without male exposure. We used laparoscopy to examine ovaries for corpora lutea and measured serum progesterone concentrations. Six of the seven isolated females failed to ovulate, while seven of the eight females exposed to males produced one to four corpora lutea. Furthermore, isolated females had significantly lower progesterone concentrations than females exposed to males. Thus, our data suggest that the American black bear is an induced ovulator. These results may aid biologists in their efforts to reproduce ursids in controlled environments.     

Evidence that hemorrhagic hypotension is mediated by the ventrolateral periaqueductal gray region

Severe hemorrhage lowers arterial pressure by suppressing sympathetic activity. This study tested the hypothesis that the decompensatory phase of hemorrhage is mediated by the ventrolateral periaqueductal gray (vlPAG), a region importantly involved in the autonomic and behavioral responses to stress and trauma. Neuronal activity in the vlPAG was inhibited with either lidocaine or cobalt chloride 5 min before hemorrhage (2.5 ml/100 g body wt) was initiated in conscious, unrestrained rats. Bilateral injection of lidocaine (0.5 microl of a 2% or 1 microl of a 5% solution) into the caudal vlPAG delayed the onset and reduced the magnitude of the hypotension produced by hemorrhage significantly. In contrast, inactivation of the dorsolateral PAG with lidocaine was ineffective. Cobalt chloride (5 mM; 0.5 microl), which inhibits synaptic transmission but not axonal conductance, also attenuated hemorrhagic hypotension significantly. Microinjection of lidocaine or cobalt chloride into the vlPAG of normotensive, nonhemorrhaged rats did not influence cardiovascular function. These data indicate that the vlPAG plays an important role in the response to hemorrhage.