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Intestinal reactive hyperemia is an abrupt blood flow increase following release from anterior mesenteric arterial occlusion. We investigated the role of adenosine in reactive hyperemia. In anesthetized rats, mesenteric arterial velocity of blood flow was determined with pulsed Doppler velocimetry and arterial pressure with a transducer. Three indices quantifying reactive hyperemias obtained following 30, 60, and 120 s arterial occlusions included duration, the volume of blood flow exceeding preocclusion blood flow, and the percentage increase in conductance. In six rat groups (half fasted and half with intrajejunal bile-oleate solutions), hyperemia parameters were determined before and after administration of either adenosine deaminase (ADA) or two adenosine receptor antagonists, namely 8-phenyltheophylline (8-PT) and 1,3-dipropyl-7-methylxanthine (DPMX). In fasted gut the three agents had variable effectiveness against reactive hyperemia, although 8-PT was the most consistent inhibitor. Instillation of intrajejunal lipid evoked a stable hyperemia and increased duration and blood flow volume after each occlusive period. ADA and 8-PT were more effective against reactive hyperemia in fed gut than in fasted gut. Our findings suggest that adenosine is a vasodilator metabolite modulating mesenteric reactive hyperemia, especially during enhanced intestinal metabolic activity.

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We investigated mechanisms mediating bradykinin (BK)-induced anterior mesenteric artery (AMA) vasodilation in anesthetized rats. The velocity of blood flowing (VBF) in the AMA was measured with pulsed Doppler velocimetry, and arterial pressure (BP) was measured with a pressure transducer. Drugs were infused through an intra-aortic catheter positioned proximal to the AMA origin. AMA conductance (C) was calculated from mean VBF/BP and expressed as percent of control C. BK infusion (10-1,000 ng.kg-1.min-1) increased C significantly (Cmax = 201 +/- 18%, ED50 = 100 ng.kg-1.min-1, P < 0.01 for all doses). A B2-subtype receptor antagonist, D-Arg,[Hyp3,Thi5.8,D-Phe7]BK, administered at 10(5) ng.kg-1.min-1 before or during BK infusion, inhibited the vasodilation by 73 +/- 7 and 103 +/- 7%, respectively. A nitric oxide (NO) synthesis inhibitor, NG-nitro-L-arginine, administered at 5.0 mg/kg 15 min before BK, inhibited the hyperemia by 61 +/- 8%. Neither a B1-receptor antagonist nor intrajejunal capsaicin inhibited BK-induced vasodilation. BK-evoked, dose-dependent, mesenteric vasodilation in rats appears to be mediated partly by B2-receptors and endogenous NO generation.

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This study was designed to evaluate the role of adenosine and adenosine receptors in the reactive (RH) and functional hyperemia (FH) in rat gut. Experiments were performed on anesthetized rats. Mesenteric blood flow was measured with a pulsed Doppler flowmeter. We also determined the duration of reactive hyperemia, excess volume of blood flow above control value and maximal increase in mesenteric vascular conductance during both hyperemic responses. Data were collected following release from occlusions lasting 30, 60 and 120 sec. Functional hyperemia was induced by perfusion of the gut with a solution. Studied parameters were obtain before and after adenosine deaminase (ADA) and two adenosine receptor antagonists: 8-phenyltheophylline (8-PT) and 1.3-dipropyl-7-methyl-xanthine (DPMX). In fasted rats ADA and 8-PT reduced of RH after each period of occlusion and DPMX was ineffective in reducing any parameter of RH. In fed rats control mesenteric blood flow was increased. ADA, 8-PT, and DPMX were more effective inhibitors of RH and FH. Above findings suggest that adenosine play a role in the modulation of RH and FH acting on A2 subtype receptors.

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We examined the specificity of a bovine hypothalamic neurosecretory granule enzyme which we discovered and which is capable of processing pro-gonadotropin releasing hormone precursor protein to yield gonadotropin associated peptide and a C-terminal extended form of gonadotropin releasing hormone (Palen et al.). The sequence in the precursor protein that separates the two active peptides is -Gly-Gly-Lys-Arg- where the pair of basic residues, -Lys-Arg-, is the anticipated cleavage site. On the basis of Vmax/Km as the measure of substrate specificity, Benzoyl(Bz)-Gly-Gly-Lys-Arg-2-Napthylamide (NA) greater than Bz-Gly-Gly-Arg-Lys-2-NA much greater than Bz-Gly-Gly-Arg-Arg-2-NA approximately equal to Bz-Gly-Gly-Lys-Lys-2-NA. Bz-Gly-Gly-Lys(N(epsilon)-acetyl)-Arg-2-NA is a very poor substrate. Our results indicate that the composition and sequence of the pair of basic residues at the primary cleavage site is important for enzyme specificity and that changes in the P1 or P2 residues of a potential substrate may affect both Km and Vmax. Hydrolysis of all substrates occurs at the P1-2-NA bond. We had previously shown that there is no cleavage between the pair of basic residues. With longer peptide substrates, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA greater than Bz-Gly-Leu-Arg(NO2)-Pro-Gly-Gly-Lys-Arg-2-NA greater than Bz-Gly-Gly-Lys-Arg-2-NA. Extending the substrate sequence to more closely resemble the amino acid sequence in the precursor protein improves Km 10-fold and Vmax about 5-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

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A new membrane bound protease has been identified in bovine hypothalamic neurosecretory granules using synthetic substrates that we prepared based on the sequence in pro-gonadotropin-releasing hormone protein that overlaps gonadotropin-releasing hormone and gonadotropin-associated peptide (thought to be prolactin-releasing hormone-inhibiting hormone). The enzyme was solubilized from neurosecretory granules using the detergent Triton X-100 and was further purified by high-performance gel permeation liquid chromatography. The enzyme hydrolyzes the Arg-2-naphthylamide (NA) bond of benzoyl(Bz)-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA which contains two likely processing sites, Arg-Pro and Lys-Arg. On the basis of the ratio of Vmax to Km as a measure of substrate specificity, Bz-Gly-Leu-Arg-Pro-Gly-Gly-Lys-Arg-2-NA is about 50-fold better than Bz-Gly-Gly-Lys-Arg-2-NA. Bz-Leu-Arg-2-NA and Bz-Gly-Leu-Arg-Pro-Gly-Gly are not hydrolyzed. The pH optimum for hydrolysis is 7.2 (Bz-Gly-Gly-Lys-Arg-2-NA substrate). As determined by gel permeation chromatography, the apparent molecular weight of the enzyme depends on the chromatography conditions; in the absence of NaCl, the Mr is approximately equal to 160,000 but is approximately equal to 80,000 if NaCl is included in the eluting buffer. After high-performance gel permeation liquid chromatography, the peak fraction containing the enzyme was lyophilized and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis; silver staining revealed a single protein band, Mr approximately equal to 70,000.

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The chromatin structure of regulatory regions of the extrachromosomal rRNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome.

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The chromatin structure of the palindromic macronuclear ribosomal RNA genes of Tetrahymena thermophila was probed with micrococcal nuclease. Independent of the state of transcriptional activity, the transcribed region had a shorter nucleosome repeat (184 +/- 3 base pairs) than the non-transcribed central spacer or bulk chromatin (both 200 base pairs). The transcribed region displayed an increased sensitivity to micrococcal nuclease in rapidly growing cells, which suggested an altered chromatin structure during transcription. At early stages of nuclease digestion, the central spacer appeared to be in a highly structured nucleosomal array. Based on the differences in nucleosome repeat distance and sensitivity to nuclease, we conclude that quite different chromatin structures are maintained in two adjacent regions of the Tetrahymena ribosomal RNA gene. The DNA of the non-transcribed terminal spacer was found to contain sequences which are highly susceptible to micrococcal nuclease, precluding any conclusions about nucleosome structure in this region.

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DNA renaturation kinetics was used to examine the relative accessibility of various regions of the Tetrahymena ribosomal RNA gene (rDNA) chromatin to micrococcal nuclease. In nuclei from cells active in rRNA transcription, the transcribed region of the rDNA chromatin was as much as 5-fold more accessible than the average of the total chromatin. As few as 20% inactive genes in the population could have accounted for all of the hybridization, so the transcribed region of the active units may be totally unprotected from nuclease degradation. The terminal non-transcribed spacer downstream from the transcription unit was also preferentially digested, but to a smaller degree. The central non-transcribed spacer was degraded to the same extent as total chromatin after a high degree of nuclease digestion. In nuclei from starved cells, which have 96% reduced rRNA transcription, the transcribed and terminal spacer regions of the rDNA were again more accessible than the total chromatin from the same nuclei, but the difference did not exceed 2-fold. We conclude that transcriptional activation is accompanied by major changes in the structure of the ribosomal gene chromatin, and that the extent and/or type of structural alteration differs in each functionally defined region of the rDNA.

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