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G Kunos

Publications and source records attributed to G Kunos.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cannabinoid-induced hypotension and bradycardia in rats mediated by CB1-like cannabinoid receptors.

Previous studies indicate that the CB1 cannabinoid receptor antagonist, N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-met hyl-1H-pyrazole-3-carboxamide HCl (SR141716A), inhibits the anandamide- and delta9-tetrahydrocannabinol- (THC) induced hypotension and bradycardia in anesthetized rats with a potency similar to that observed for SR141716A antagonism of THC-induced neurobehavioral effects. To further test the role of CB1 receptors in the cardiovascular effects of cannabinoids, we examined two additional criteria for receptor-specific interactions: the rank order of potency of agonists and stereoselectivity. A series of cannabinoid analogs including the enantiomeric pair (-)-11-OH-delta9-THC dimethylheptyl (+)-11-OH-delta9-THC dimethylheptyl were evaluated for their effects on arterial blood pressure and heart rate in urethane anesthetized rats. Six analogs elicited pronounced and long lasting hypotension and bradycardia that were blocked by 3 mg/kg of SR141716A. The rank order of potency was (-)-11-OH-delta9-THC dimethylheptyl > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol > THC > anandamide > or = (-)-3-[2-hydroxy-4-(1,1-dimethyl-heptyl)phenyl]-4-[3-hydroxy-propyl]c yclohexan-1-ol, which correlated well with CB1 receptor affinity or analgesic potency (r = 0.96-0.99). There was no hypotension or bradycardia after palmitoylethanolamine or (+)-11-OH-delta9-THC dimethylheptyl. An initial pressor response was also observed with THC and anandamide, which was not antagonized by SR141716A. We conclude that the similar rank orders of potency, stereoselectivity and sensitivity to blockade by SR141716A indicate the involvement of CB1-like receptors in the hypotensive and bradycardic actions of cannabinoids, whereas the mechanism of the pressor effect of THC and anandamide remains unclear.

Animals↗
PubMed1997

Cardiovascular effects of ethanol in rats selectively bred for high or low sensitivity to the hypnotic effects of ethanol.

The cardiovascular effects of acute ethanol administration were studied in rats selectively bred for high (HAS) or low sensitivity (LAS) to the acute hypnotic effect of ethanol. In chronically cannulated, conscious animals, the intravenous administration of 1 g/kg of ethanol caused moderate hypotension and tachycardia in both HAS and LAS rats. The phenylephrine-induced reflex bradycardia and the baroreflex slope were significantly reduced by ethanol only in HAS and not in LAS rats. In urethane-anesthetized HAS rats, the baroreflex inhibitory action of ethanol was present under control conditions, but it was absent after depletion of endogenous GABA by treatment of the rats with 100 mg/kg of 3-mercaptopropionate. These findings confirm the role of endogenous GABA in the baroreflex inhibitory action of ethanol, and are compatible with the documented differential sensitivity of the GABAA, receptor complex to ethanol in HAS and LAS rats.

Animals↗
PubMed1996

DNA elements and protein factors involved in the transcription of the beta 2-adrenergic receptor gene in rat liver. The negative regulatory role of C/EBP alpha.

Primer extension and RNase protection analyses of the rat beta 2-adrenergic receptor (beta 2AR) gene identify two transcription start points at -64 and -220 nt, respectively. Transient transfections of putative promoter/pCAT constructs into DDT1 MF-2 cells indicate that fragments -36 to -100 (PI) and -186 to -312 (P2) are sufficient to promote transcription, whereas -911 to -1122 contains a negative regulatory element(s). RNase protection analysis of the 3' untranslated region (3'-UTR) indicates the presence of two transcripts with 3'-UTR of 111 and 604 nt exclusive of the poly(A+) tails. Northern blots of beta 2AR mRNA using full-length and partial cDNA probes indicate that a major 2.2 kb and a minor 1.6 kb species arise from the use of alternative promoters as well as different polyadenylation signals. DNase I footprinting and DNA mobility shift assays (DMSA) using rat liver nuclear extracts identify a number of transcription factors binding to sequence elements within or upstream from P1 and P2, including Spl, CRE, CPl, AP-2, NF-1, NF-kappa B, and C/EBP. Supershift assays using antibodies against C/EBP alpha and C/EBP beta and mutational analyses indicate that the protein binding to the C/EBP consensus recognition site at -925 to -933 is C/EBP alpha. The activity of promoter/CAT constructs containing the C/EBP recognition site is significantly decreased by cotransfection of C/EBP alpha but not C/EBP alpha but not C/EBP beta into either DDT1 MF-2 cells or primary rat hepatocytes. Partial hepatectomy causes a transient decrease in C/EBP alpha, as measured by DMSA, and an increase in beta 2 AR mRNA levels and rate of transcription in the remnant liver. Thus, derepression via C/EBP alpha is likely involved in the up-regulation of beta 2AR in the regenerating rat liver.

Animals↗
PubMed1996

Ethanol inhibition of stress-related tachycardia involves medullary NMDA receptors.

In rats, neurons in the perifornical area of the hypothalamus send descending projections to the commissural part of the nucleus tractus solitarii as demonstrated by an anterograde tracer study. In urethane-anaesthetised rats, stimulation of neurons in the perifornical area by microinjection of bicuculline or 6-OH-saclofen causes tachycardia and inhibits baroreflex bradycardia. The effects elicited from the perifornical area are similar in magnitude to those elicited from the adjacent dorsomedial nucleus, also called the hypothalamic defense area. Microinjection into the nucleus tractus solitarii of the NMDA (N-methyl-D-aspartate) receptor antagonist, AP-7 (2-amino-7-phosphonoheptanoic acid), inhibits the tachycardic response to stimulation of the perifornical area. Injection of ethanol intravenously or into the nucleus tractus solitarii also inhibits this tachycardic response, but causes no further inhibition when combined with AP-7. We conclude that the perifornical area is part of the hypothalamic defense area, and it is under strong, tonic GABAergic inhibition mediated by both GABAA and GABAB receptors. Furthermore, descending input from the perifornical area to the nucleus tractus solitarii is via an NMDA synapse, and ethanol inhibits stress-related tachycardia by inhibiting these NMDA receptors in the nucleus tractus solitarii.

Animals↗
PubMed1996

Melanocortin antagonists define two distinct pathways of cardiovascular control by alpha- and gamma-melanocyte-stimulating hormones.

Melanocortin peptides and at least two subtypes of melanocortin receptors (MC3-R and MC4-R) are present in brain regions involved in cardiovascular regulation. In urethane-anesthetized rats, unilateral microinjection of alpha-melanocyte-stimulating hormone (MSH) into the medullary dorsal-vagal complex (DVC) causes dose-dependent (125-250 pmol) hypotension and bradycardia, whereas gamma-MSH is less effective. The effects of alpha-MSH are inhibited by microinjection to the same site of the novel MG4-R/MC3-R antagonist SHU9119 (2-100 pmol) but not naloxone (270 pmol), whereas the similar effects of intra-DVC injection of beta-endorphin (1 pmol) are inhibited by naloxone and not by SHU9119. Hypotensive and bradycardic responses to electrical stimulation of the arcuate nucleus also are inhibited by ipsilateral intra-DVC microinjection of SHU9119. gamma-MSH and ACTH(4-10), but not alpha-MSH, elicit dose-dependent (0.1-12.5 nmol) pressor and tachycardic effects, which are much more pronounced after intracarotid than after intravenous administration. The effects of gamma-MSH (1.25 nmol) are not inhibited by the intracarotid injection of SHU9119 (1.25-12.5 nmol) or the novel MC3-R antagonist SHU9005 (1.25-12.5 nmol). We conclude that the hypotension and bradycardia elicited by the release of alpha-MSH from arcuate neurons is mediated by neural melanocortin receptors (MC4-R/MC3-R) located in the DVC, whereas the similar effects of beta-endorphin, a peptide derived from the same precursor, are mediated by opiate receptors at the same site. In contrast, neither MC3-R nor MC4-R is involved in the centrally mediated pressor and tachycardic actions of gamma-MSH, which, likely, are mediated by an as yet unidentified receptor.

Animals↗
PubMed1996

Inhibition of exocytotic noradrenaline release by presynaptic cannabinoid CB1 receptors on peripheral sympathetic nerves.

1. Activation of CB1 receptors by plant cannabinoids or the endogenous ligand, anandamide, causes hypotension via a sympathoinhibitory action in anaesthetized rats. In mouse isolated vas deferens, activation of CB1 receptors inhibits the electrically evoked twitch response. To determine if these effects are related to presynaptic inhibition of noradrenaline (NA) release, we examined the effects of delta 9-tetrahydrocannabinol (delta 9-THC), anandamide and the CB1 antagonist, SR141716A, on exocytotic NA release in rat isolated atria and vasa deferentia. 2. In isolated atria and vasa deferentia preloaded with [3H]-NA, electrical field stimulation caused [3H]-NA release, which was abolished by tetrodotoxin 0.5 microM and concentration-dependently inhibited by delta 9-THC or anandamide, 0.3-10 microM. The inhibitory effect of delta 9-THC and anandamide was competitively antagonized by SR 141716A, 1-10 microM. 3. Tyramine, 1 microM, also induced [3H]-NA release, which was unaffected by tetrodotoxin, delta 9-THC or anandamide in either atria or vasa deferentia. 4. CB1 receptor mRNA is present in the superior cervical ganglion, as well as in whole brain, cerebellum, hypothalamus, spleen, and vas deferens and absent in medulla oblongata and atria, as demonstrated by reverse transcription-polymerase chain reaction. There was no evidence of the presence of CB1A receptor mRNA in ganglia, brain, or cerebellum. These results suggest that activation of presynaptic CB1 receptors located on peripheral sympathetic nerve terminals mediate sympathoinhibitory effects in vitro and in vivo.

Animals↗
PubMed1996

Transcriptional regulation of alpha(1b) adrenergic receptors (alpha(1b)AR) by nuclear factor 1 (NF1): a decline in the concentration of NF1 correlates with the downregulation of alpha(1b)AR gene expression in regenerating liver.

The 5' upstream region from --490 to --540 (footprint II) within the dominant P2 promoter of the rat alpha(1b) adrenergic receptor (alpha(1b)AR) gene is recognized by a sequence-specific DNA-binding protein (B. Gao, M. S. Spector, and G. Kunos, J. Biol. Chem. 270:5614-5619, 1995). This protein, detectable in Southwestern (DNA-protein) blots of crude nuclear extracts as 32- and 34-kDa bands, has been purified 6,000-fold from rat livers by DEAE-Sepharose, heparin-Sepharose, and DNA affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and UV cross-linking of the purified protein indicated the same molecular mass as that in crude extracts. Methylation interference analysis revealed strong contact with a TTGGCT hexamer and weak contact with a TGGCGT hexamer in the 3' and 5' portions of footprint II, respectively. Nucleotide substitutions within these hexamers significantly reduced protein binding to footprint II and the promoter activity of P2 in Hep3B cells. The purified protein also bound to the nuclear factor 1 (NF1)/CTF consensus sequence, albeit with lower affinity. Gel mobility supershift and Western blotting (immunoblotting) analyses using an antibody against the NF1/CTF protein identified the purified 32- and 34-kDa polypeptides as NF1 or a related protein. Cotransfection into Hep3B cells or primary rat hepatocytes of cDNAs of the NF1-like proteins NF1/L, NF1/X, and NF1/Redl resulted in a three- to fivefold increase in transcription directed by wild-type P2 but not by the mutated P2. Partial hepatectomy markedly decreased the levels of NF1 in the remnant liver and its binding to P2, which paralleled declines in the rate of transcription of the alpha(1b)AR gene and in the steady-state levels of its mRNA. These observations indicate that NF1 activates transcription of the rat alpha(1b)AR gene via interacting with its P2 promoter and that a decline in the expression of NF1 is one of the mechanisms responsible for the reduced expression of the alpha(1b)AR gene during liver regeneration.

Animals↗
PubMed1996

alpha-2-Adrenergic activation of proopiomelanocortin-containing neurons in the arcuate nucleus causes opioid-mediated hypotension and bradycardia.

Treatment of rats for 4 days with alpha-methyldopa, 200 mg/kg/day i.p., increases steady state levels of proopiomelanocortin (POMC) mRNA in the mediobasal hypothalamus, as measured by DNA excess solution hybridization. The increase is prevented by parallel treatment with yohimbine, 2 mg/kg/day i.p., but not by naltrexone, 2 mg/kg/day i.p. Treatment with the peripheral vasodilator hydralazine, 2 mg/kg/day, does not affect POMC mRNA levels. In situ hybridization histochemistry with a cRNA probe for POMC indicates that POMC-containing cells are located within the confines of the arcuate nucleus both in control and in alpha-methyldopa-treated rats, and confirms the increase in POMC mRNA in the latter. Microinjection of 2 micrograms of alpha-methylnorepinephrine unilaterally into the arcuate nucleus of urethane-anesthetized rats causes hypotension and bradycardia, which can be inhibited by 200 ng of yohimbine microinjected into the same site, or by 100 ng l-naloxone microinjected into the ipsilateral nucleus tractus solitarii, but not into the arcuate nucleus. These findings are interpreted to indicate that activation of alpha 2-adrenergic receptors located on POMC-containing neurons in the arcuate nucleus causes beta-endorphin release and stimulation of opiate receptors in the NTS, which results in hypotension and bradycardia, and that this mechanism contributes to the hypotensive action of alpha-methyldopa.

Adrenergic alpha-Agonists↗
PubMed1996

Mechanism of the hypotensive action of anandamide in anesthetized rats.

We studied the effects of the endogenous cannabinoid ligand anandamide on blood pressure, single unit activity of barosensitive neurons in the rostral ventrolateral medulla, and postganglionic splanchnic sympathetic nerve discharge in urethane-anesthetized rats. In rats with an intact baroreflex, an intravenous bolus of 4 mg/kg anandamide caused a triphasic blood pressure response: transient hypotension, followed by a brief pressor and more prolonged depressor phase. Anandamide evoked a "primary" increase in neuronal firing coincident with its pressor effect and a "secondary," baroreflex-mediated rise coincident with its depressor effect at both sites. Pretreatment of rats with phentolamine or trimethaphan did not inhibit either the pressor response or the primary increase in splanchnic nerve discharge elicited by anandamide. In barodenervated rats, electrical stimulation of the rostral ventrolateral medulla increased blood pressure and splanchnic nerve discharge. Anandamide treatment blunted the rise in blood pressure without affecting the increase in splanchnic nerve discharge. Anandamide did not affect the rise in blood pressure in response to an intravenous bolus dose of phenylephrine. The results indicate that (1) the brief pressor response to anandamide is not sympathetically mediated, and (2) the prolonged hypotensive response to anandamide is not initiated in the central nervous system, in ganglia, or at postsynaptic adrenergic receptors but is due to a presynaptic action that inhibits norepinephrine release from sympathetic nerve terminals in the heart and vasculature.

Anesthesia↗
PubMed1995

Sequence of the 5' regulatory domain of the gene encoding the rat beta 2-adrenergic receptor.

Restriction mapping and sequence analysis of the gene encoding the rat beta 2 adrenergic receptor (beta 2AR) revealed an error affecting most of the 5'-flanking domain, located between -192 and -2245 bp in the only published sequence of this gene [Buckland et al., Nucleic Acids Res. 18 (1990) 682]. The correct sequence lacks a TATA box, contains 67% G + C residues in the first 500 nucleotides upstream from the start codon, and contains putative glucocorticoid, thyroid hormone and cAMP-response elements.

Animals↗
PubMed1995

Novel antagonist implicates the CB1 cannabinoid receptor in the hypotensive action of anandamide.

In anaesthetised rats, the endogenous cannabinoid anandamide has potent cardiovascular effects that include a brief pressor effect and a more prolonged depressor response. The depressor response is attenuated after transection of the cervical spinal cord or blockade of alpha-adrenergic receptors by phentolamine, and is dose-dependently inhibited by a selective antagonist of the CB1 cannabinoid receptor. The pressor component is not affected by any of these interventions. This suggests that the depressor response is due to inhibition of sympathetic tone mediated by CB1 receptors, whereas the pressor component is due to a peripheral action that does not involve the same receptors or the sympathetic nervous system.

Anesthesia↗
PubMed1995

The rat alpha 1B adrenergic receptor gene middle promoter contains multiple binding sites for sequence-specific proteins including a novel ubiquitous transcription factor.

Transcription of the rat alpha 1B adrenergic receptor (alpha 1BAR) gene in the liver is controlled by three promoters that generate three mRNAs. The middle promoter (P2), located between -432 and -813 base pairs upstream from the translation start codon and lacking a TATA box, is responsible for generating the major, 2.7-kilobase mRNA-species expressed in many tissues (Gao, B., and Kunos, G. (1994) J. Biol. Chem. 269, 15762-15767). DNase I footprinting using rat liver nuclear extracts identified three protected regions in P2: footprint I (-432 to -452), footprint II (-490 to -540), and footprint III (-609 to -690). Putative response elements in footprints I and III were not analyzed except the AP2 binding site in footprint III, which could be protected by purified AP2 protein. Footprint II contains four sites corresponding to half of the NF-I consensus sequence, but DNA mobility shift assays indicate that this footprint binds two proteins distinct from NF-I: a ubiquitous CP1-related factor and another novel factor, termed alpha-Adrenergic Receptor Transcription Factor (alpha ARTF), which binds to two separate sites in this region. The alpha ARTF is widely distributed, with the highest amounts found in brain, followed by liver, kidney, lung, and spleen, but no detectable activity in heart. Deletions of alpha ARTF binding sites nearly abolished P2 promoter activity, which suggests that the alpha ARTF is essential for the transcription of the alpha 1BAR gene in most tissues.

Animals↗
PubMed1994

Transcription of the rat alpha 1B adrenergic receptor gene in liver is controlled by three promoters.

The proximal 5'-flanking region of the rat alpha 1B adrenergic receptor (alpha 1BAR) gene contains discrete transcription start points (tsp) utilized in liver, located at -54, -57 (tsp1), and -443 base pairs (tsp2) upstream from the translation start codon (Gao, B., and Kunos, G. (1993) Gene (Amst.) 131, 243-247). Primer extension analyses using 5' upstream primers now identify an additional cluster of tsp between -1035 and -1340 base pairs (tsp3). Northern blots of rat liver mRNA reveal three alpha 1BAR mRNAs of 2.3, 2.7, and 3.3 kilobases in length. Transient transfections of putative promoter/pCAT constructs document the existence of three promoters, P1 (-127, -49), P2- (-813, -432), and P3 (-1363, -1107), which direct transcription from tsp1, tsp2, and tsp3, respectively. P1 contains no recognition sequences for known transcription factors. P2 is (G + C)-rich, lacks a TATA box, and contains a cAMP response element, GC, CACC, and GCAAT boxes, and binding sites for nuclear factor I. P3 contains a putative TATATA and CCAAT box and is flanked by recognition sites for the liver-specific CCAAT/enhancer binding protein and hepatocyte nuclear factor 5. These findings indicate that heterogeneity of alpha 1BAR mRNA in liver is related to transcription of the gene by three distinct promoters. Differential control of these promoters may underlie the well documented developmental and tissue-specific regulation of the alpha 1BAR.

Animals↗
PubMed1994

Endogenous gamma-aminobutyric acid (GABA) mediates ethanol inhibition of vagally mediated reflex bradycardia elicited from aortic baroreceptors.

We have previously demonstrated that ethanol depresses baroreflex bradycardia by potentiating the similar action of endogenous gamma-aminobutyric acid (GABA) in the medullary dorsal vagal complex. In the present study we examined the relative contribution of the sympathetic vs. the parasympathetic nervous system and aortic vs. carotid sinus baroreceptors in this effect. Depressor baroreflex responses were elicited in urethane-anesthetized male Sprague-Dawley rats by i.v. injection of graded bolus doses of phenylephrine or by electrical stimulation of the aortic nerve at different frequencies. Methyl-atropine (2 mg/kg i.v.) greatly attenuated, and bilateral cervical vagotomy completely eliminated, phenylephrine-induced reflex bradycardia, whereas propranolol (1 mg/kg i.v.) caused a moderate decrease in the reflex bradycardic response. Ethanol (1 g/kg i.v) did not influence the residual reflex bradycardia after methyl-atropine, but significantly decreased the residual reflex bradycardia after propranolol. Aortic nerve stimulation caused frequency-dependent hypotension, which was unaffected by methyl-atropine, and bradycardia, which was eliminated by methyl-atropine. Depletion of endogenous GABA by pretreatment of rats with 3-mercaptopropionate slightly increased the bradycardic response to aortic nerve stimulation and eliminated its susceptibility to inhibition by ethanol. Acute aortic nerve denervation moderately reduced the reflex bradycardic response to phenylephrine, which was no longer sensitive to inhibition by ethanol. These findings suggest that 1) ethanol inhibits baroreflex bradycardia but not hypotension, 2) the effect of ethanol is selective regarding both the afferent (aortic vs. carotid baroreceptors) and efferent limbs of the reflex (vagal vs. sympathetic) and 3) the effect of ethanol is mediated through endogenous GABA, probably at the level of the dorsal brainstem.

Animals↗
PubMed1993

Isolation and characterization of the gene encoding the rat alpha 1B adrenergic receptor.

Using a rat alpha 1B adrenergic receptor(AR)-encoding cDNA probe, we isolated two genomic clones from a rat liver genomic DNA library. Southern blot analysis and nucleotide sequencing indicate that the rat alpha 1B AR gene has two exons and a single large intron of at least 16 kb. Analysis of the sequence of the 5'-flanking region suggests that this gene has the features of a housekeeping gene: it has neither a TATA box nor a CAAT box, but has multiple transcription start points (tsp), multiple Sp1-binding sites, and a high G + C content. The 5'-flanking region also contains consensus sequences for AP1- and AP2-binding sites, and putative sites for cyclic AMP (CRE), glucocorticoid (GRE), and thyroid responses (TRE). The 3'-flanking region contains a putative polyadenylation signal (ATTAAA) 492 bp downstream from the stop codon. A comparison of the rat alpha 1B AR sequence with that of the recently cloned human alpha 1B AR gene showed that the overall structure of this gene is highly conserved, with some notable differences in the 5'-flanking region.

Animals↗
PubMed1993

Sequence of a cDNA encoding bovine apolipoprotein H.

The nucleotide sequence of the ApoH cDNA encoding the bovine apolipoprotein H (ApoH) has been determined. The deduced protein, which contains a 19-amino-acid (aa) signal peptide and the 326-aa mature ApoH, shows 89% and 86% homology with human and rat ApoH, respectively.

Amino Acid Sequence↗