Dopamine receptor modulation of noradrenaline release from renal sympathetic nerves was investigated. Human kidney slices were incubated with 3H-noradrenaline, placed into superfusion chambers between two platinum electrodes and field-stimulated at 5 Hz. The slices accumulated radioactivity. Pretreatment of the kidney slices with 6-hydroxy-dopamine (1.2 mM) prior to the 3H-noradrenaline incubation reduced the accumulation of radioactivity. The stimulation induced (S-I) outflow of radioactivity was mainly composed of intact 3H-noradrenaline. The sodium channel blocker tetrodotoxin (1 microM), 6-hydroxy-dopamine pretreatment and omission of calcium from the superfusion solution abolished S-I outflow of radioactivity. The DA1-receptor agonist fenoldopam (SKF 82526; 0.01 and 0.1 microM) did not alter but fenoldopam (1 microM) increased S-I outflow of radioactivity. However, in the presence of either the non-selective alpha-adrenoceptor antagonist phentolamine (1 microM) or the selective alpha 2-adrenoceptor antagonist idazoxan (1 microM) fenoldopam (1 microM) had no effect. The DA2-receptor agonist quinpirole (LY 171555; 1 microM) inhibited S-I outflow of radioactivity, an effect blocked by the selective DA2-receptor antagonists S(-)-sulpiride (10 microM) and domperidone (0.3 microM) but unaltered either by the DA1-receptor antagonist SCH 23390 (1 microM) or by phentolamine (1 microM). The alpha 2-adrenoceptor agonist UK 14304 (0.1 microM) inhibited S-I outflow of radioactivity, and this effect was blocked by phentolamine (1 microM) and idazoxan (1 microM) but unaltered by S(-)-sulpiride (10 microM). Phentolamine and idazoxan, in contrast to S(-)-sulpiride, domperidone and SCH 23390, enhanced S-I outflow of radioactivity by themselves.(ABSTRACT TRUNCATED AT 250 WORDS)
Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid-polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10(3) overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10(1)-10(2) by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.
Rat kidney slices were incubated with [3H]-noradrenaline and placed into a superfusion chamber between two platinum electrodes. The kidney slices accumulated and stored radioactivity. In kidney slices taken from rats whose sympathetic nerve terminals were destroyed by pretreatment with 6-hydroxydopamine accumulation of radioactivity was abolished. The a2-adrenoceptor antagonist idazoxan (0.1-1 F.M) enhanced but tetrodotoxin (TTX, 1 ,LM) or omission of calcium from the superfusion solution abolished the stimulation induced (S-I) outflow of radioactivity. Angiotensin (A) I (3-300 nM) and AII (1-100 nM) enhanced S-I outflow of radioactivity. The effect of AI was markedly attenuated by the angiotensin converting enzyme inhibitor captopril (3 FLM) and that of AII was blocked by the AII receptor antagonist saralasin (1 FLM). These results suggest that the kidney slice preparation is a valid technique to study modulation of renal noradrenaline release. Endogenous noradrenaline released from sympathetic nerves in rat kidney slices activates prejunctional a2-adrenoceptors to inhibit its own release. AII, which can also be formed locally from AI in these kidney slices, activates prejunctional AII receptors to facilitate renal noradrenaline release.
The effect of the dopamine D2-receptor agonist carmoxirole on noradrenaline release was investigated in human and rat cortical kidney slices. After preincubation with 3H-noradrenaline, the slices were electrically stimulated at 5 Hz in superfusion chambers, and the stimulation-induced (S-I) outflow of radioactivity was taken as the index of noradrenaline release. In human but not in rat cortical kidney slices, carmoxirole (0.03 microM) inhibited the S-I outflow of radioactivity. Carmoxirole (0.3 microM) also failed to inhibit the S-I outflow of radioactivity from human kidney slices. When alpha-adrenoceptors were blocked by the non-selective alpha-adrenoceptor antagonist phentolamine (1 microM), carmoxirole (0.03 microM, 0.3 microM) inhibited S-I outflow to a similar extent. The inhibitory effect of carmoxirole (0.03 microM) was prevented by the D2-receptor antagonist (-)-sulpiride (10 microM) but not by the D1-receptor antagonist SCH 23390 (1 microM) in human kidney slices. Phentolamine (1 microM) by itself induced a five-fold greater enhancement of the S-I outflow of radioactivity in rat than in human cortical kidney slices. The data suggest that activation of prejunctional D2-receptors by carmoxirole inhibits noradrenaline release from human renal sympathetic nerves. Carmoxirole in higher concentrations (0.3 microM) blocks inhibitory prejunctional alpha-autoreceptors, which seems to mask the inhibitory D2-receptor mediated effect. The different effects of phentolamine and carmoxirole in human and rat kidney may indicate a difference of the prejunctional alpha-autoreceptor mechanism in the two species.
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