Differential distribution of functional alpha(1)-adrenergic receptor subtypes along the rat tail artery

The rat tail artery has been used for the study of vasoconstriction mediated by alpha(1A)-adrenoceptors (ARs). However, rings from proximal segments of the tail artery (within the initial 4 cm, PRTA) were at least 3- fold more sensitive to methoxamine and phenylephrine (n = 6 - 12; p < 0.05) than rings from distal parts (between the sixth and 10th cm, DRTA). Interestingly, the imidazolines N-[ 5-( 4,5- dihydro- 1H- imidazol-2-yl)-2-hydroxy-5,6,7,8- tetrahydronaphthalen- 1- yl] methanesulfonamide hydrobromide (A-61603) and oxymetazoline, which activate selectively alpha(1A)- ARs, were equipotent in PRTA and DRTA (n = 4 - 12), whereas buspirone, which activates selectively alpha(1D)-AR, was approximate to 70-fold more potent in PRTA than in DRTA (n = 8; p < 0.05). The selective alpha(1D)-AR antagonist 8-[2-[4-(methoxyphenyl)-1-piperazinyl] ethyl]-8-azaspiro[4.5] decane-7,9-dione dihydrochloride (BMY- 7378) was approximate to 70- fold more potent against the contractions induced by phenylephrine in PRTA (pK(B) of approximate to 8.45; n = 6) than in DRTA (pK B of approximate to 6.58; n = 6), although the antagonism was complex in PRTA. 5-Methylurapidil, a selective alpha(1A)-antagonist, was equipotent in PRTA and DRTA (pK(B) of approximate to 8.4), but the Schild slope in DRTA was 0.73 +/- 0.05 ( n = 5). The noncompetitive alpha(1B)-antagonist conotoxin rho-TIA reduced the maximal contraction induced by phenylephrine in DRTA, but not in PRTA. These results indicate a predominant role for alpha(1A)-ARs in the contractions of both PRTA and DRTA but with significant coparticipations of alpha(1D)-ARs in PRTA and alpha(1B)-ARs in DRTA. Semiquantitative reverse transcription-polymerase chain reaction revealed that mRNA encoding alpha(1A)- and alpha(1B)-ARs are similarly distributed in PRTA and DRTA, whereas mRNA for alpha(1D)-ARs is twice more abundant in PRTA. Therefore, alpha(1)-ARs subtypes are differentially distributed along the tail artery. It is important to consider the segment from which the tissue preparation is taken to avoid misinterpretations on receptor mechanisms and drug selectivities. antagonism was complex in PRTA. 5- Methylurapidil, a selective alpha(1A)-antagonist, was equipotent in PRTA and DRTA (pK(B) of approximate to 8.4), but the Schild slope in DRTA was 0.73 +/- 0.05 ( n = 5). The noncompetitive alpha(1B)-antagonist conotoxin rho-TIA reduced the maximal contraction induced by phenylephrine in DRTA, but not in PRTA. These results indicate a predominant role for alpha(1A)-ARs in the contractions of both PRTA and DRTA but with significant coparticipations of alpha(1D)-ARs in PRTA and alpha(1B)-ARs in DRTA. Semiquantitative reverse transcription-polymerase chain reaction revealed that mRNA encoding alpha(1A)- and alpha(1B)- ARs are similarly distributed in PRTA and DRTA, whereas mRNA for alpha(1D)-ARs is twice more abundant in PRTA. Therefore, alpha(1)-ARs subtypes are differentially distributed along the tail artery. It is important to consider the segment from which the tissue preparation is taken to avoid misinterpretations on receptor mechanisms and drug selectivities.

A Modified Technique of Rat Tail Suspension for Longer Periods of Observation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

alpha(1)-Adrenoceptors in proximal segments of tail arteries from control and reserpinised rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

SEX-RELATED DIFFERENCES IN THE ANALGESIC RESPONSE TO THE RAT TAIL IMMERSION TEST

The analgesic response was evaluated by the tail immersion test in adult male (N = 30), female (N = 21) and androgenized female Wistar rats (N = 15). The reaction time for tail withdrawal from the hot water bath was faster for male than for female rats (3.48 +/- 0.12 vs 6.46 +/- 0.42 s). The reaction time of androgenized female rats was similar to that of male rats (3.08 +/- 0.16 s). Blockade of opiate receptors with naloxone (2 mg/kg, ip) decreased the sensitivity to the noxious stimuli in males (4.08 +/- 0.10 s) and in androgenized females (3.69 +/- 0.19 s) but increased it in female rats (5.01 +/- 0.41 s). These data show sex-related differences in the analgesic response evaluated by the tail immersion test and indicate that administration of androgens to newborn female rats affects their pain sensitivity.

Influence of physical preconditioning on the responsiveness of rat pulmonary artery after pulmonary ischemia/reperfusion

The aim of this work was to evaluate the effect of physical preconditioning in the responsiveness of rat pulmonary rings submitted to lung ischemia/reperfusion (IR). Wistar rats were divided into three groups: Sedentary sham-operated (SD/SHAM); sedentary submitted to ischemia/reperfusion (SD/IR) and trained submitted to ischemia/reperfusion (TR/IR) animals. Exercise training consisted in sessions of 60 min/day running sessions, 5 days/week for 8 weeks. Left pulmonary IR was performed by occluding for 90 min and reperfusing for 120 min. After that, pulmonary arteries were isolated and concentration-response curves to acetylcholine (ACh), histamine (HIST), sodium nitroprusside (SNP), phenylephrine and U46619 were obtained. Neither potency (- log EC50) nor maximal responses (Emax) were modified for ACh and HIST in all groups. On the other hand, the potency for SNP was significantly increased in TR/IR group (8.23 ± 0.06) compared to SD/IR group (7.85 ± 0.04). Contractile responses mediated by a-adrenergic receptor were markedly decreased in IR groups (SD/IR: 6.75 ± 0.06 and TR/IR: 6.62 ± 0.04) compared to SD/SHAM (7.33 ± 0.05). No changes were seen for the U46619 in all groups. In conclusion, the present study shows that exercise training has no protective actions in the local blood vessel where the IR process takes place. © 2006 Elsevier Inc. All rights reserved.

Differential phosphorylation, desensitization, and internalization of α1A-adrenoceptors activated by norepinephrine and oxymetazoline

Loss of response on repetitive drug exposure (i.e., tachyphylaxis) is a particular problem for the vasoconstrictor effects of medications containing oxymetazoline (OXY), an α1-adrenoceptor (AR) agonist of the imidazoline class. One cause of tachyphylaxis is receptor desensitization, usually accompanied by phosphorylation and internalization. It is well established that a1A-ARs are less phosphorylated, desensitized, and internalized on exposure to the phenethylamines norepinephrine (NE), epinephrine, or phenylephrine (PE) than are the a1B and a1D subtypes. However, here we show in human embryonic kidney-293 cells that the low-efficacy agonist OXY induces G protein-coupled receptor kinase 2-dependent a1A-AR phosphorylation, followed by rapid desensitization and internalization (∼40% internalization after 5 minutes of stimulation), whereas phosphorylation of α1A-ARs exposed to NE depends to a large extent on protein kinase C activity and is not followed by desensitization, and the receptors undergo delayed internalization (∼35% after 60 minutes of stimulation). Native α1A-ARs from rat tail artery and vas deferens are also desensitized by OXY, but not by NE or PE, indicating that thisproperty of OXY is not limited to recombinant receptors expressed in cell systems. The results of the present study are clearly indicative of agonist-directed a1A-AR regulation. OXY shows functional selectivity relative to NE and PE at a1A-ARs, leading to significant receptor desensitization and internalization, which is important in view of the therapeutic vasoconstrictor effects of this drug and the varied biologic process regulated by α1A-ARs. Copyright © 2013 by The American Society for Pharmacology and Experimental Therapeutics.

Fosforilação, dessensibilização e internalização de adrenoceptores α1A ativados por noradrenalina e oximetazolina: participação diferencial da PKC e da GRK2

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effects of crude extracts of Agaricus blazei on DNA damage and on rat liver carcinogenesis induced by diethylnitrosamine

The effects of crude extracts of the mushroom Agaricus blazei Murrill (Agaricaceae) on both DNA damage and placental form glutathione S-transferase (GST-P)-positive liver foci induced by diethylnitrosamine (DEN) were investigated. Six groups of adult male Wistar rats were used. For two weeks, animals of groups 3 to 6 were treated with three aqueous solutions of A. blazei (mean dry weight of solids being 1.2, 5.6, 11.5 and 11.5 mg/ml, respectively). After this period, groups 2 to 5 were given a single ip injection 200 mg/kg DEN and groups 1 and 6 were treated with 0.9% NaCl. All animals were subjected to 70% partial hepatectomy at week five and sacrificed 4, 24 and 48 h or 8 weeks after DEN or 0.9% NaCl treatments (10th week after the beginning of the experiment). The alkaline comet assay and GST-P-positive liver foci development were used to evaluate the influence of the mushroom extracts on liver cell DNA damage and on the initiation of liver carcinogenesis, respectively. Previous treatment with the highest concentration of A. blazei (11.5 mg/ml) significantly reduced DNA damage, indicating a protective effect against DEN-induced liver cytotoxicity/genotoxicity. However, the same dose of mushroom extract significantly increased the number of GST-P-positive liver foci.

Estudo das propriedades analgésicas da Achillea millefolium L. (Asteraceae)

This study was designed to separately evaluate the analgesic capacity of 15% tea from Achillea millefolium leaves and florid ends. Three different tests were performed. The animals were separated in 3 groups: group I (control) that received water, group II that received florid end tea, and group III that received leaf tea. The first test was the writhing test induced by intraperitoneal injection of acetic acid in male Swiss mice. The second test consisted of the measurement of tail flick reflex latency (TFRL) in male Wistar rats after receiving a painful thermal stimulus. The groups received the teas or water per os, prior to observation. In the third test, the Wistar rats were submitted to an approximately 1 cm long incision in their tails to test the effect of the teas on induced inflammatory pain. The results showed that the effect of the 15% leaf tea on the mice writhing and TFRL test were not statistically significant. The florid ends tea showed a significantly increase in number of writhes in mice and TFRL in rats after administration. With regard to the effect of the teas on inflammatory pain, ingestion of both preparations caused an increase in the force threshold necessary for the avoidance reflex, indicating antihyperalgesic action of both the leaf and florid ends 15% teas of Achillea millefolium.

Interação entre as vias de sinalização de receptores serotoninérgicos e Β-adrenérgicos em artéria femoral de ratos

Fundamento: A doença coronária tem sido amplamente estudada em pesquisas cardiovasculares. No entanto, pacientes com doença arterial periférica (DAP) têm piores resultados em comparação àqueles com doença arterial coronariana. Portanto, os estudos farmacológicos com artéria femoral são altamente relevantes para a melhor compreensão das respostas clínicas e fisiopatológicas da DAP. Objetivo: Avaliar as propriedades farmacológicas dos agentes contráteis e relaxantes na artéria femoral de ratos. Métodos: As curvas de resposta de concentração à fenilefrina contrátil (FC) e à serotonina (5-HT) e os agentes relaxantes isoproterenol (ISO) e forskolina foram obtidos na artéria femoral de ratos isolada. Para as respostas ao relaxamento, os tecidos foram contraídos com FC ou 5-HT. Resultados: A potência de classificação na artéria femoral foi de 5-HT > FC para as respostas contráteis. Em tecidos contraídos com 5-HT, as respostas de relaxamento ao isoproterenol foram praticamente abolidas em comparação aos tecidos contraídos com FC. A forskolina, um estimulante da adenilil ciclase, restaurou parcialmente a resposta de relaxamento ao ISO em tecidos contraídos com 5-HT. Conclusão: Ocorre uma interação entre as vias de sinalização dos receptores β-adrenérgicos e serotoninérgicos na artéria femoral. Além disso, esta pesquisa fornece um novo modelo para estudar as vias de sinalização serotoninérgicas em condições normais e patológicas que podem ajudar a compreender os resultados clínicos na DAP.

Inside-out vein graft and inside-out artery graft in rat sciatic nerve repair

Although veins and arteries present similar wall structures, there are differences which may be relevant in peripheral nerve reconstruction. Inside-out vein grafts (IOVG) have been satisfactorily used to repair both motor and sensitive nerves. However, the inside-out artery graft (IOAG) is a new technique and not fully investigated. Our study presents comparative morphological data on nerve regeneration achieved with IOVG and IOAG in the repair of Wistar rat sciatic nerves. Jugular veins and aorta arteries were harvested from donor animals and used inside-out to bridge a 10-mm gap. Animals were sacrificed at 10 weeks to evaluate nerve regeneration. Both techniques presented great variability in nervous tissue, though some animals showed satisfactory results. Different intensities of scarring processes might have interfered with nerve regeneration. Although IOVG and IOAG techniques showed similar morphometric results, in general, IOVG presented a closer-to-normal nerve organization than IOAG.

Age related obesity-induced shortening of GLUT4 mRNA poly(A) tail length in rat gastrocnemius skeletal muscle

Obese insulin resistant animals and humans have shown reduced GLUT4 gene expression. Yet, in skeletal muscle, discrepancy between mRNA and protein regulation has been frequently observed, suggesting a post-transcriptional modulation. We investigated the GLUT4 expression in adipose tissue and muscle of obese 12-month-old (12-mo) rats, comparing with lean 2-month-old (2-mo) animals. Obesity was accompanied by insulin resistance, and 65% reduction (P < 0.01) in GLUT4 mRNA and protein in adipose tissue. However, in muscle, despite increased (P < 0.05) mRNA content, GLUT4 protein was unchanged. RNase H and poly(A) test assays showed a reduction (P < 0.01) of ∼80 adenines in the GLUT4 mRNA poly(A) tail of muscle from 12-mo rats, recognizing that the poly(A) tail length correlates with translation efficiency. Concluding, age related obesity of 12-mo rats involves suppression of GLUT4 expression in adipose tissue; however, in muscle, GLUT4 mRNA content increases, but with a shorter poly(A) tail, thus unchanging the protein content. © 2007 Elsevier B.V. All rights reserved.

Evaluation of level of DNA damage in blood leukocytes of non-diabetic and diabetic rat exposed to cigarette smoke

The objective of the present study was to use the comet assay to evaluate the steady-state level of DNA damage in peripheral blood leukocytes from diabetic and non-diabetic female Wistar rats exposed to air or to cigarette smoke. A total of 20 rats were distributed into four experimental groups (n= 5 rats/group): non-diabetic (control) and diabetic exposed to filtered air; non-diabetic and diabetic exposed to cigarette smoke. A pancreatic beta (beta)-cytotoxic agent, streptozotocin (40 mg/kg b.w.) was used to induce experimental diabetes in rats. Rats placed into whole-body exposure chambers were exposed for 30 min to filtered air (control) or to tobacco smoke generated from 10 cigarettes, twice a day, for 2 months. At the end of the 2-month exposure period, each rat was anesthetized and humanely killed to obtain blood samples for genotoxicity analysis using the alkaline comet assay. Blood wleukocytes sampled from diabetic rats presented higher DNA damage values (tail moment =0.57 +/- 0.05; tail length =19.92 +/- 0.41, p < 0.05) compared to control rats (tail moment =0.34 +/- 0.02; tail length= 17.42 +/- 0.33). Non-diabetic (tail moment =0.43 +/- 0.04, p > 0.05) and diabetic rats (tail moment= 0.41 +/- 0.03, p > 0.05) exposed to cigarette smoke presented non-significant increases in DNA damage levels compared to control group. In conclusion, our data show that the exposure of diabetic rats to cigarette smoke produced no additional genotoxicity in peripheral blood cells of female Wistar rats. (c) 2007 Elsevier B.V. All rights reserved.

Hipertensão arterial experimental e prenhez em ratas: uso do modelo Goldblatt I (1 rim - 1 clipe)

Objetivo: desenvolver o modelo experimental de hipertensão tipo Goldblatt I (1 rim - 1 clipe), em ratas, para estudar a interação entre hipertensão e prenhez. Métodos: o experimento foi dividido em 5 períodos: adaptação (2 semanas), cirúrgico (1 semana), desenvolvimento da hipertensão (6 semanas), acasalamento e estabilização da pressão arterial (6 semanas) e prenhez (3 semanas). Foram utilizadas 82 ratas virgens da raça Wistar, pesando entre 180-240 gramas e com idade entre 3 e 4 meses. As ratas foram sorteadas para compor os 4 grupos experimentais (controle, manipulação, nefrectomia e hipertensão) e estudadas em 15 momentos distintos (M1 a M15). A hipertensão foi induzida experimentalmente pela técnica de Goldblatt I (1 rim, 1 clipe), que consiste na constrição da artéria renal esquerda e nefrectomia contralateral. Posteriormente, foram realizadas medidas periódicas da pressão arterial pelo método da pletismografia de cauda (PAC). Resultados: os animais sem tratamento cirúrgico (controle) e com manipulação não apresentaram alterações na PAC durante o experimento. A nefrectomia determinou discreta elevação da PAC. Nos grupos de ratas prenhes, observou-se tendência a discreta diminuição da PAC, que se acentuou no final da prenhez. Conclusões: o modelo experimental foi adequado para o objetivo de nosso estudo, pois permitiu a obtenção de animais hipertensos.