Biomedical subjects

G Kunos

Publications and source records attributed to G Kunos.

At least 19 recordsLinked to original sources

PubMed1998

Effects of ethanol on mitogen-activated protein kinase and stress-activated protein kinase cascades in normal and regenerating liver.

To understand the mechanisms by which ethanol inhibits hepatocyte proliferation, we studied the effects of ethanol on p42/44 mitogen-activated protein kinase (MAPK), p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK) in normal and regenerating rat liver. Treatment of rat hepatocytes with 100 mM ethanol in vitro for 16 h prolonged the activation of p42/44 MAPK and p38 MAPK induced by various agonists. Such treatment also increased basal JNK activity, but did not potentiate or prolong agonist-induced JNK activation. Ethanol potentiation of the activation of p42/44 MAPK was abolished by pertussis toxin. In contrast, chronic ethanol consumption in vivo inhibited the activation of p42/44 MAPK, p38 MAPK and JNK induced either by partial hepatectomy or by various agonists. However, both acute and chronic ethanol inhibited hepatocyte proliferation induced by insulin and epidermal growth factor. A selective inhibitor of p42/44 MAPK partially prevented the inhibition of hepatocyte proliferation caused by acute, but not by chronic, ethanol exposure, whereas a selective inhibitor of p38 MAPK further inhibited hepatocyte proliferation under both conditions. These data suggest that acute and chronic ethanol inhibit hepatocyte proliferation by different mechanisms. The effect of acute ethanol may be related to the prolongation of p42/44 MAPK activation, whereas inhibition of hepatocyte proliferation by chronic ethanol may be due to inhibition of p38 MAPK activation.

Alcoholism↗

PubMed1998

Effects of intracellular calcium on GABAA receptors in mouse cortical neurons.

Using the patch-clamp technique, we studied the effect of intracellular Ca2+ on Cl- current gated by type A gamma-aminobutyric acid receptors (GABAA) in mouse cortical neurons. When the rapid Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) was in the pipette solution, the GABA-activated Cl- current amplitude decreased over time to 49 +/- 7% of control. In contrast, equimolar replacement of BAPTA with ethylenebis(oxonitrilo)tetraacetate (EGTA) caused a 60 +/- 10% increase in GABA current. An increased intracellular Ca2+ concentration caused a transient augmentation of the GABA current. This effect of Ca2+ was concentration dependent (10 nM to 34 muM). Ca2+ increased the amplitude of the current by enhancing the maximal response to GABA rather than by changing the affinity of the receptor to GABA (EC50 = 5 +/- 0.4 muM vs. 7 +/- 0.3 muM). Both calmodulin (CaM) and a CaM kinase II inhibitor (200 muM) blocked the potentiating effect of Ca2+ suggesting that it was mediated by activation of CaM kinase II. We found that regulation of GABAA receptors by intracellular Ca2+ in cortical neurons has important physiological implications since the potentiating effect of increasing the intracellular Ca2+ on responses to GABA was mimicked by activating excitatory receptors with 100 muM N-methyl-D-aspartate (NMDA). These findings suggest that modulation of GABAA receptor activity by glutamate may be brought about via changes in intracellular Ca2+.

Animals↗

PubMed1998

Histone H1 isoforms purified from rat liver bind nonspecifically to the nuclear factor 1 recognition sequence and serve as generalized transcriptional repressors.

Two polypeptides with molecular masses of 34 and 30 kDa were copurified from rat liver during DNA affinity purification of a sequence-specific transcription factor binding to the footprint II sequence within the P2 promoter of the rat alpha1B adrenergic receptor (alpha1B AR) gene, and were identified by microsequencing their endoproteinase Lys-C-derived peptides as histone H1d and histone H1c, respectively. Histone H1 was previously reported to bind to the nuclear factor 1 (NF1) recognition sequence, although the specificity of this binding has been controversial. Here, DNA mobility shift and supershift assays, DNase I footprinting and mutational analyses indicated that the binding of histone H1 to the NF1 sites located within footprint II of the alpha1B AR gene P2 promoter is nonspecific. Transient cotransfections into Hep3B cells of histone H1d cDNA with CAT constructs containing promoter regions of different genes resulted in generalized and non-specific suppression of CAT activity. The histone H1d-mediated repression of the activities of the alpha1B AR gene P2/CAT or beta2 AR gene P(-186/1307)/CAT constructs was reversed by the cotransfection of a cDNA encoding the sequence-specific transcription factor NF1/X, and the fold increase in CAT activities was similar to that obtained in the absence of histone H1d. These results suggest that sequence-specific transcription factors counteract the histone H1-mediated transcriptional repression in vivo by a true activation, which is different from the in vitro antirepression in histone H1-repressed chromatin templates (Laybourn and Kadonaga, (1991) Science 254: 238-245).

Animals↗

PubMed1998

Platelet- and macrophage-derived endogenous cannabinoids are involved in endotoxin-induced hypotension.

Macrophages are the primary cellular targets of bacterial lipopolysaccharide (LPS), but the role of macrophage-derived cytokines in LPS-induced septic shock is uncertain. Recent evidence indicates that activation of peripheral CB1 cannabinoid receptors contributes to hemorrhagic hypotension and that macrophage-derived anandamide as well as unidentified platelet-derived substances may be contributing factors. Here we demonstrate that rat platelets contain the endogenous cannabinoid 2-arachidonyl glyceride (2-AG), as identified by reverse phase high-performance liquid chromatography, gas chromatography, and mass spectrometry, and that in vitro exposure of platelets to LPS (200 microg/ml) markedly increases 2-AG levels. LPS-stimulated, but not control, macrophages contain anandamide, which is undetectable in either control or LPS-stimulated platelets. Prolonged hypotension and tachycardia are elicited in urethane-anesthetized rats treated 1) with LPS (15 mg/kg i.v.); 2) with macrophages plus platelets isolated from 3 ml of blood from an LPS-treated donor rat; or 3) with rat macrophages or 4) platelets preincubated in vitro with LPS (200 microg/ml). In all four cases, the hypotension but not the tachycardia is prevented by pretreatment of the recipient rat with the CB1 receptor antagonist SR141716A (3 mg/kg i.v.), which also inhibits the hypotensive response to anandamide or 2-AG. The hypotension elicited by LPS-treated macrophages or platelets remains unchanged in the absence of sympathetic tone or after blockade of nitric oxide synthase. These findings indicate that platelets and macrophages generate different endogenous cannabinoids, and that both 2-AG and anandamide may be paracrine mediators of endotoxin-induced hypotension via activation of vascular CB1 receptors.

Animals↗

PubMed1997

Activation of peripheral CB1 cannabinoid receptors in haemorrhagic shock.

Anandamide, an endogenous cannabinoid ligand, binds to CB1 cannabinoid receptors in the brain and mimics the neurobehavioural actions of marijuana. Cannabinoids and anandamide also elicit hypotension mediated by peripheral CB1 receptors. Here we report that a selective CB1 receptor antagonist, SR141716A, elicits an increase in blood pressure in rats subjected to haemorrhagic shock, whereas similar treatment of normotensive rats or intracerebroventricular administration of the antagonist during shock do not affect blood pressure. Blood from haemorrhaged rats causes hypotension in normal rats, which can be prevented by SR141716A but not by inhibition of nitric oxide synthase in the recipient. Macrophages and platelets from haemorrhaged rats elicit CB1 receptor-mediated hypotension in normotensive recipients, and incorporate arachidonic acid or ethanolamine into a product that co-elutes with anandamide on reverse-phase high-performance liquid chromatography. Also, macrophages from control rats stimulated with ionomycin or bacterial phospholipase D produce anandamide, as identified by gas chromatography and mass spectrometry. These findings indicate that activation of peripheral CB1 cannabinoid receptors contributes to haemorrhagic hypotension, and anandamide produced by macrophages may be a mediator of this effect.

Animals↗

PubMed1997

Effects of short and long term ethanol on the activation of signal transducer and activator transcription factor 3 in normal and regenerating liver.

Interleukin-6 (IL-6) induced activation of Signal Transducer and Activator Transcription Factor 3 (Stat3) is a critical step in liver regeneration. Chronic ethanol consumption is known to increase the plasma concentration of IL-6, yet the ability of the liver to regenerate and the regenerative induction of several IL-6 initiated events are impaired in chronic alcoholic liver disease. We hypothesized that chronic ethanol consumption inhibits IL-6 dependent signal transduction. To test this hypothesis, the effect of ethanol on the Stat3 signal transduction pathway was studied in the adult rat liver. In vitro treatment of freshly isolated normal adult rat hepatocytes with 50-100 mM ethanol for 30 min blocked IL-6-induced Stat3 activation. Long-term ethanol intake in vivo significantly attenuated the activation of Stat3 induced either in vivo by partial hepatectomy or in vitro by IL-6. In contrast, short-term ethanol consumption enhanced the regenerative induction of Stat3 but inhibited IL-6 induced Stat3 activation. These data suggest that the inhibition of liver regeneration by chronic ethanol consumption is, at least in part, mediated by modulating the activation of Stat3.

Administration, Oral↗

PubMed1997

Sp1-mediated transcriptional activation from the dominant promoter of the rat alpha1B adrenergic receptor gene in DDT1MF-2 cells.

In the rat liver, NF1 and CP1 bind to the major P2 promoter of the alpha1B adrenergic receptor gene to generate footprint II. Here we show that, in DDT1MF-2 smooth muscle cells, the major protein bound to footprint II is not NF1 but Sp1, which binds to the 5'-portion of the footprint II sequence (footprint IIb). Mutational analyses demonstrate that the CCCGCG sequence in footprint IIb is critical for Sp1 binding and P2 promoter activity. A second GC box in the P2 promoter also binds the Sp1 protein and contributes to the P2 promoter activity. Gel shift assays indicate that footprint II can bind Sp1, NF1, and CP1, and that the binding of these 3 proteins is mutually exclusive. This is also indicated by the results of functional cotransfection experiments, where transient overexpression of NF1 and Sp1 together caused a similar increase in the activity of a P2/CAT reporter construct as overexpression of either Sp1 or NF1 alone, indicating lack of additivity. The preferential interaction of footprint II with Sp1 in DDT1MF-2 cells and NF1 in liver appears to be due to low levels of NF1 expression in DDT1MF-2 cells and low levels of Sp1 in liver. These observations suggest that NF1 and Sp1 are the major transcription factors involved in controlling the P2 promoter in liver versus DDT1MF-2 cells, respectively, which may be one of the mechanisms responsible for the complex tissue-specific regulation of the expression of the alpha1B adrenergic receptor gene.

Animals↗

PubMed1997

Positive and negative regulation of JNK1 by protein kinase C and p42(MAP kinase) in adult rat hepatocytes.

The role of protein kinase C (PKC) and p42(MAP kinase) signaling in the regulation of proliferation and apoptosis was investigated in freshly isolated and primary cultured rat hepatocytes. Acute treatment of freshly isolated hepatocytes with phenylephrine and EGF caused rapid phasic activations of p42(MAP kinase) and JNK1. Acute pre-treatment of hepatocytes with the PKC inhibitors sphingosine, chelerythrine and bis-indolylmaleimide abolished the ability of phenylephrine, but not EGF, to activate p42(MAP kinase) and JNK1. Acute pretreatments with all of the PKC inhibitors alone increased JNK1 basal activity approximately 2-fold. Acute treatments of primary cultures of hepatocytes with an inhibitor of MEK1 activation (PD98059) also caused inhibition of p42(MAP kinase) and a approximately 2-fold activation of JNK1. These data demonstrate that PKC can function as both a proximal activator and a distal inhibitor of signaling through the JNK1/SAP kinase pathway. Treatments (4 h) of primary cultured hepatocytes with sphingosine, chelerythrine, bis-indolylmaleimide and PD98059 did not induce apoptosis as judged by propidium iodide staining. Similar acute treatments of HepG2 cells rapidly induced cell death. These data demonstrate that acute inhibition of either PKC or p42(MAP kinase) function is sufficient to rapidly induce apoptosis in transformed, but not in non-transformed hepatocytes.

Animals↗

PubMed1997

Both the cyclic AMP response element and the activator protein 2 binding site mediate basal and cyclic AMP-induced transcription from the dominant promoter of the rat alpha 1B-adrenergic receptor gene in DDT1MF-2 cells.

cAMP markedly increases alpha 1B adrenergic receptor (alpha 1B-AR) expression in FRTL-5 and PC C13 rat thyroid cells, DDT1MF-2 smooth muscle cells, primary rat hepatocytes, and K9 rat liver cells. Here, we used DDT1MF-2 cells to evaluate further the mechanisms by which cAMP stimulates alpha 1B-AR expression. Receptor binding assays, Northern blotting, and nuclear run-on analyses demonstrated that forskolin (1 microM) in the presence of isobutylmethylxanthine (0.25 mM) increased alpha 1B-AR numbers, mRNA level, and gene transcription rate by 2.3 +/- 0.2-, 2.5 +/- 0.3-, and 3.5 +/- 0.2-fold over control, respectively. Dibutyryl cAMP (1 mM) plus isobutylmethylxanthine (0.25 mM) also enhanced alpha 1B-AR density by 2.7 +/- 0.1-fold over control. Further experiments demonstrated that the induction of alpha 1B-AR by forskolin requires new protein synthesis and is protein kinase A dependent. In DDT1MF-2 cells transfected with alpha 1B-AR gene P2 promoter/CAT constructs, both forskolin and dibutyryl cAMP significantly increased P2 promoter activity. The P2 promoter region of the rat alpha 1B-AR gene (-813 to -432) contains a cAMP response element (CRE) (-444 to -437) and an AP2 binding site (-647 to -638). Mutations in either one of these elements alone led to a decrease in both basal and cAMP-induced P2 promoter activity. Mutations in both elements caused a further inhibition of basal transcription and a complete block of cAMP-induced P2 promoter activity. Direct binding of purified activator protein 2 (AP2) to the AP2 element in the P2 promoter was reported previously. Gel mobility shift and super-shift assays using liver nuclear extracts from either rat liver or DDT1MF-2 cells demonstrated that the CRE in the alpha 1B-AR gene bound CRE binding protein. These data indicate that both the CRE and the AP2 element in the P2 promoter contribute to basal as well as cAMP-induced transcription of the alpha 1B-AR gene in DDT1MF-2 cells.

Animals↗

PubMed1997

Differential regulation of the mitogen-activated protein and stress-activated protein kinase cascades by adrenergic agonists in quiescent and regenerating adult rat hepatocytes.

To study the mechanisms by which catecholamines regulate hepatocyte proliferation after partial hepatectomy (PHX), hepatocytes were isolated from adult male rats 24 h after sham operation or two-thirds PHX and treated with catecholamines and other agonists. In freshly isolated sham cells, p42 mitogen-activated protein (MAP) kinase activity was stimulated by the alpha1-adrenergic agonist phenylephrine (PHE). Activation of p42 MAP kinase by growth factors was blunted by pretreatment of sham hepatocytes with glucagon but not by that with the beta2-adrenergic agonist isoproterenol (ISO). In PHX cells, the ability of PHE to activate p42 MAP kinase was dramatically reduced, whereas ISO became competent to inhibit p42 MAP kinase activation. PHE treatment of sham but not PHX and ISO treatment of PHX but not sham hepatocytes also activated the stress-activated protein (SAP) kinases p46/54 SAP kinase and p38 SAP kinase. These data demonstrate that an alpha1- to beta2-adrenergic receptor switch occurs upon PHX and results in an increase in SAP kinase versus MAP kinase signaling by catecholamines. In primary cultures of hepatocytes, ISO treatment of PHX but not sham cells inhibited [3H]thymidine incorporation. In contrast, PHE treatment of sham but not PHX cells stimulated [3H]thymidine incorporation, which was reduced by approximately 25 and approximately 95% with specific inhibitors of p42 MAP kinase and p38 SAP kinase function, respectively. Inhibition of the p38 SAP kinase also dramatically reduced basal [3H]thymidine incorporation. These data suggest that p38 SAP kinase plays a permissive role in liver regeneration. Alterations in the abilities of catecholamines to modulate the activities of protein kinase A and the MAP and SAP kinase pathways may represent one physiological mechanism by which these agonists can regulate hepatocyte proliferation after PHX.

Adrenergic Agonists↗

PubMed1997

Cardiovascular effects of anandamide in anesthetized and conscious normotensive and hypertensive rats.

We previously showed that in anesthetized rats anandamide elicits bradycardia and a triphasic blood pressure response: transient hypotension secondary to a vagally mediated bradycardia, followed by a brief pressor and prolonged depressor response, the latter two effects being similar to those of delta 9-tetrahydrocannabinol (THC). The prolonged depressor but not the pressor response was reduced after alpha-adrenergic receptor blockade or cervical spinal cord transection and was inhibited by the cannabinoid type 1 (CB1) receptor antagonist SR141716A, suggesting CB1 receptor-mediated sympathoinhibition as the underlying mechanism. Here we examined the relationship between sympathetic tone and the cardiovascular effects of anandamide by testing these effects in both conscious and anesthetized, normotensive and spontaneously hypertensive rats. In urethane-anesthetized normotensive rats, SR141716A inhibited the prolonged depressor and bradycardic effects of anandamide and THC with similar potency, whereas it did not affect the pressor response to either agent. Anadamide caused similar hypotension in spontaneously breathing and in paralyzed, mechanically ventilated rats, suggesting that the hypotension is not secondary to respiratory effects. In conscious normotensive rats, anandamide elicited transient vagal activation and a brief pressor response, but the prolonged hypotensive component was absent. SR141716A potentiated and prolonged the brief pressor response to anandamide, suggesting that the depressor response may have been masked by an increased pressor response. All three phases of the anadamide response were present in both anesthetized and conscious spontaneously hypertensive rats, and the hypotensive component, inhibited by SR141716A in both, was more prolonged in the absence (> 50 minutes) than the presence (10 to 15 minutes) of anesthesia. We conclude that anandamide causes a non-CB1 receptor-mediated pressor and a CB1 receptor-mediated prolonged depressor response. The depressor response can be elicited in both conscious and anesthetized animals, but its magnitude depends on preexisting sympathetic tone.

Anesthesia↗

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↗