Search

To avoid some of the disadvantages associated with using radiolabeling to investigate adenyl purine content and release from excitable tissues, a reverse-phase high-pressure liquid chromatographic method utilizing fluorescence detection for the measurement of picomole amounts of endogenous ATP and its 6-amino purine analogs has been developed. This procedure has been used to determine the content of adenyl purines in the guinea pig vas deferens and that released from the tissue following stimulation of adrenergic nerves. The total tissue content was measured to be 1.6, 0.76, and 0.10 mumol/g of ATP, ADP, and AMP, respectively. However, adenosine could not be detected. Hypoxia caused a significant decrease in ATP content concomitant with an increase in adenosine content to 0.04 mumol/g. Following transmural electrical stimulation of the guniea pig vas deferens, the release of the following purine compounds was detected: ATP (0.106 nmol/g), ADP (0.242 nmol/g), AMP (0.035 nmol/g), and adenosine (0.454 nmol/g).

The histamine content in plasma samples obtained from a group of healthy blood donors, from blood stored in citrate-phosphate-dextrose supplemented with adenine (CPDA) packs for up 28 days, and from the side arm of a transfusion line was measured by radio-enzymatic assay. Healthy donors had a mean plasma histamine content of 0.79 ng per ml. Blood stored in CPDA initially showed a similar histamine level (0.69 ng/ml on day 3 of storage), but there was a progressive rise with time, and at 28 days, the level was 20.5 ng per ml. The increase in histamine is best described by a positive exponential and may be explained by a process whereby the plasma histamine level increases the degree of histamine release from blood cells. The histamine levels in the blood infused into patients tended to be higher than those found in the stored units of the same age, if these packs were less than 7 days old. This may have been caused by the unit becoming warmer during transfusion. We speculate that the histamine levels in the older units of stored blood were high enough to cause or augment transfusion reactions and that the storage age of blood may have a bearing on the incidence of transfusion reactions.

The change in norepinephrine (NE) content with age (from 2 days to 17 weeks old) was examined in a variety of tissues from spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. NE content was determined by either a catechol-O-methyltransferase-based radioenzymatic assay or high performance liquid chromatography with electrochemical detection. Regardless of the age of the animal, NE content per gram of tissue was significantly greater in mesenteric arteries and kidneys from SHR compared to WKY tissues, whereas NE content per whole kidney was similar between the two rat strains. The time course of enhanced NE content in caudal arteries and aortas from SHR followed the development of hypertension. In the spleen, NE content per gram of tissue was elevated in young SHR; however, in adult rats NE content was not significantly different between the two rat strains. Because spleens from WKY rats were substantially larger, total NE content per spleen was significantly greater in tissues from WKY rats. Cardiac contents of NE were similar in SHR and WKY rats at all ages examined. Adrenal epinephrine concentrations were similar in SHR and WKY rats, whereas NE content was elevated in the SHR at 46 and 81 days of age. The results of the present study demonstrate that the appearance of increased NE levels in some SHR tissues occurs before the development of hypertension in this model. If NE content is a valid index of sympathetic innervation, enhanced innervation may contribute to the vascular medial hypertrophy observed in young SHR and the elevation of blood pressure in these rats.

Three semi-automated procedures are described for the estimation of the catecholamine contents of urine, tissue and plasma samples. The three procedures are based on the fluorometric tri-hydroxyindole assay which has been modified for automatic analysis. These techniques offer several advantages over currently available assays in that they are more convenient, provide for faster analysis rate and give increased sensitivity. The results of the present studies in which the catecholamine contents of urine, tissue and plasma samples were determined using the semi-automated assay provided a range of values which was identical to that obtained using other methods.

A sensitive automated assay was developed for the determination of histamine. The endogenous histamine content of intact rabbit thoracic aorta, and of its separated adventitial and medial layers was determined. The histamine content of the intact aorta was found to be only partially sensitive to compound 48/80. The isolated aorta accumulated labelled histamine against a concentration gradient. The uptake was abolished by cooling to 1 degree, but was unaffected by oxygen lack. The uptake process was sensitive to a variety of agents including extraneuronal catecholamine uptake inhibitors (deoxycorticosterone acetate, normetanephrine) as well as inhibitors of catecholamine and histamine metabolism (tranylcypromine, hydrallazine). The uptake was not affected by amodiaquin, a histamine N-methyl-transferase inhibitor, by the metabolites 1,4-methylhistamine or imidazole acetic acid, by compound 48/80 or by the basic amino acid arginine.

In the present study the influence of oestradiol, catechol oestrogens, and O-methylated oestrogens was determined on the contractile responses of the isolated rabbit aorta to (-)-adrenaline. Oestradiol (40 mumol/l), 2-hydroxyoestradiol (2OHE2) (20 mumol/l), and 2-methoxyoestradiol (2MeOE2) (20 mumol/l) all sensitized the rabbit aorta to contractile responses to (-)-adrenaline. Similarly, the 2-hydroxy and 2-methoxy derivatives of oestrone and oestriol also sensitized the aorta to (-)-adrenaline-induced contractions. The largest degree of sensitization was seen in the presence of the 2-methoxysteroids. Oestradiol and 2OHE2 did not increase responses of the aorta to (-)-noradrenaline, while slight potentiation of contraction was seen in the presence of 2MeOE3. The potentiating effect of 2OHE2 on contractile responses to (-)-adrenaline was abolished by prior treatment of the tissue with a COMT inhibitor (U-0521, 55 mumol/l). Conversely, pretreatment of the tissue with 2OHE2 prevented the augmented aortic contraction to (-)-adrenaline usually seen after inhibition of COMT. The non-additive nature of the sensitization seen after combined treatment with 2OHE2 and U-0521 was qualitatively similar to that seen following combined exposure to maximally effective concentrations of U-0521 and an inhibitor of extraneuronal uptake (hydrocortisone 100 mumol/l). Oestradiol and 2MeOE2 reduced the formation of both the 3H-O-methylated, 3H-deaminated and the 3H-O-methylated deaminated metabolites of 3H-(-)-adrenaline (0.15 mumol/l) during exposure of the aorta to the tritiated catecholamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetes was induced in rats by a single intraperitoneal injection of streptozotocin (65 mg kg-1). Rabbits were rendered diabetic by injecting alloxan (100 mg kg-1) into the lateral ear vein. Diabetes was confirmed by a significant elevation of serum glucose in both species 8 weeks after injection. The maximum contraction to noradrenaline (NA), 5-hydroxytryptamine (5-HT) and KCl was markedly diminished in thoracic aortic rings (AR) from diabetic rats with no change in the EC50 of the agonists. There were no differences in the contractile properties of AR from diabetic rabbits to NA, 5-HT or KCl. Diabetes did not alter the responsiveness of AR from the rat to angiotensin II (AII). However, AR from diabetic rabbits displayed a decreased maximal contraction and an increased EC50 to AII. The magnitude of the acetylcholine-induced relaxation to precontracted AR was not different between diabetic and control rats and rabbits. The contractile responses of AR to NA, 5-HT and KCl were depressed in diabetic rats, regardless of the control tissue to which they were compared. The decrease in maximal contraction to NA, 5-HT and KCl seen in diabetic animals was prevented by insulin replacement. The results demonstrated that while both rats and rabbits exhibited a similar degree of hyperglycemia after treatment with a diabetogenic agent, aortic preparations from the rabbit are not affected in the same way as the aorta from the diabetic rat when exposed to NA, 5-HT and KCl. This feature may be related to the marked differences between the extent of sympathetic innervation of the aorta in the rabbit and rat. Furthermore, the decrease in maximal contraction in rat aorta was non-specific with respect to agonists since it could also be demonstrated with KCl. Therefore, it follows that the diabetic state may affect processes responsible for contraction beyond the level of receptor activation.

The use of sodium borate impregnated silica gel plates for the chromatographic separation of the catecholamines noradrenaline, adrenaline, and isoprenaline from their respective 3-0-methylated derivatives, normetanephrine, metanephrine, and methoxy-isoprenaline, is described. The parent catecholamines remain at the origin of the plates while the 3-0-methylated derivatives concentrate in discrete bands at the upper edge of the borate impregnated area (the "borate front").

When assayed on homogenates of the rabbit ear artery up to 192 h after sympathetic denervation, the activity of monoamine oxidase (MAO) showed a small but significant decrease (maximum 9%). The activity of catechol-O-methyl transferase (COMT) appeared unchanged, although it is possible that the small and variable nature of this enzyme activity compared with that of MAO may have masked a comparable effect of denervation. The maximum decreases in noradrenaline (NA) and adenosine triphosphate (ATP) contents were greater than 90% at 48 h, and 32% at 96 h after denervation. These results confirm the presence of neuronal as well as extra-neuronal MAO, and also the presence of extraneuronal comt in this artery. The decline in ATP contents after denervation suggest that part of the ATP is neuronal. However, the estimated ratio of NA to neuronal ATP is far in excess of that reported for isolated noradrenaline storage vesicles, suggesting that the major portion of the neuronal ATP was not present in these vesicles.

The influence of streptozotocin-induced diabetes on the contractile responses of the rat isolated perfused mesenteric vascular bed was determined. Two control groups were used, age-matched, fed ad-libitum rats and age-matched, food-restricted rats which were weight-matched to the diabetic group. Two months after the induction of diabetes there was a nonspecific decrease in contractile responsiveness of the mesenteric vascular bed from the diabetic rats to nerve stimulation, and to intraluminal norepinephrine and KCI compared to the responses of the control groups. There was no difference in the responsiveness of the two control groups to these vasoconstrictor treatments. The diabetes-induced depression of responses could be due to changes in vascular smooth muscle function, with an additional contribution of an abnormality in sympathetic neuronal function.

A new procedure for sympathetic denervation of the hearts of rats and guinea pigs is described. Bilateral removal of the inferior and medial cervical ganglia results in almost complete loss of catecholamines from atria and ventricles, disappearance of catecholamine-associated histofluorescence from the region of the sinoatrial node and marked depression of the chronotropic concentration-response curve for tyramine in right atria of both species. Seven days after bilateral sympathectomy, the chronotropic concentration-response curve for isoproterenol is shifted to the left by a factor of 3.3 in the rat and 1.7 in guinea-pig right atria. The chronotropic concentration-response curve for histamine was not shifted by sympathectomy in the guinea-pig right atrium. Inasmuch as the rat atrium does not respond to histamine, similar experiments could not be done in the rat. The inotropic concentration-response curve for isoproterenol in electrically driven left atria was not affected by 7 days of sympathectomy in either species. These results indicate that chronic surgical sympathectomy of the heart can be successfully accomplished in the rat and guinea pig. Such sympathectomy induces a postjunctional supersensitivity in guinea-pig right atria which is qualitatively and quantitatively similar to that described previously for chronic treatment with reserpine. Bilateral surgical sympathectomy provides a valuable tool for future investigations of the cellular basis of supersensitivity in the myocardium.

The present study examines the subcellular site of catecholamine O-methylation in extraneuronal tissue. S-Adenosyl-l-methionine, a methyl donor that does not diffuse across biological membranes, was used to assess the participation of plasma membrane bound catechol-O-methyltransferase vs. cytoplasmic catechol-O-methyltransferase in the catecholamine O-methylating process. Segments of rabbit thoracic aorta incubated with [methyl-3H]-S-adenosyl-l-methionine and isoproterenol generate [3H]methoxy-isoproterenol. The formation of [3H]methoxy-isoproterenol from [methyl-3H]-S-adenosyl-l-methionine was proportional to the isoproterenol concentrations in the range of 0.1 to 1.0 microM. There was a marked preference for the O-methylation of the (+)- rather than the (-)-isomer of isoproterenol. The O-methylation of isoproterenol in the presence of [methyl-3H]-S-adenosyl-l-methionine was stimulated as much as 8-fold by the removal of calcium ions from the incubation solutions. In contrast, the O-methylation of (+)-[3H]isoproterenol by endogenous, intracellular S-adenosyl-l-methionine was only slightly inhibited by the removal of calcium ions from incubation solutions. The formation of [3H]methoxy-isoproterenol from [methyl-3H]-S-adenosyl-l-methionine and isoproterenol was not inhibited by pretreatment of tissues with phenoxybenzamine (32 microM) or treatment with metanephrine (27 mumol 1(-1] or deoxycorticosterone acetate (27 microM), i.e., drug treatments that inhibit the extraneuronal uptake and O-methylation of [3H]-isoproterenol by endogenous intracellular S-adenosyl-l-methionine. The results of this study provide evidence for a nonintracellular, cell-associated site of O-methylation of isoproterenol in the rabbit aorta.

Factors which influence uptake on O-methylation of 3H-isoprenaline (3HISO) by isolated segments of the rabbit ear artery were investigated. The tendency of the artery to accumulate unchanged amine was enhanced either when the O-methylation of ISO approached saturation at a high substrate concentration (6 mumol 1(-1) ISO), or when O-methylation was inhibited. Under these conditions, deoxycorticosterone acetate (DOCA) partially inhibited the accumulation of ISO. DOCA exerted a stronger inhibitory effect on O-methylation, but only at low ISO concentrations (0.2 and 0.8 mumol 1(-1)). The results are interpreted as evidence that (a)O-methylation in the intact artery is a saturable process which serves to limit the accumulation of 3HISO when the bathing concentration of the amine is low, and (b) DOCA exerts its effects by inhibiting extraneuronal uptake into the compartment possessing O-methylating activity. Analysis of the kinetics of efflux of 3HISO suggested that O-methylation was restricted to only one compartment, whereas unchanged ISO was accumulated within at least two compartments in addition to the extracellular compartment. Attention is drawn to a small but significant inhibitory effect of chronic sympathetic denervation on the accumulation, although not the O-methylation of ISO, raising the possibility that there may also be distribution of 3HISO within the sympathetic nerves.

A method for the rapid preparation of parenchymal microvessels from rat, rabbit and bovine brains is described. Light and electron microscopic examination of the isolated microvessels showed the smooth muscle and endothelial cells to be intact and substantially free of neutrophil contamination. The observation of 0.2 to 2.0 micron electron dense granules in the adventitial space associated with some of the isolated arteriolar elements and the identification of histamine in microvessel extracts is suggestive of the presence of mast cells in the microvessel preparations. Age-dependent changes in histamine content and an increased histamine content in microvessels from hypertensive rats were noted. Using a sensitive radioenzymatic assay, the catecholamine (CA) contents of microvessels isolated from cow, rat and rabbit were measured. The total CA contents of all microvessel preparations were small (< 0.35 microgram g-1) with norepinephrine being the predominant CA present. A large ratio of dopamine to norepinephrine was found in microvessels from rat and bovine brain. The possible origins of the CAs present in the isolated microvessels are discussed.

The use of sodium borate impregnated thin-layer silica gel plates for the separation of noradrenaline and its 3-O-methylated metabolites is described. Its application to studies of the metabolism of tritiated I-noradrenaline by isolated tissues is illustrated for the rabbit uterus.

The levels of norepinephrine (NE), dopamine (DA) and epinephrine (E) in the vasculature of hypertension (DOCA-SALT) and normotensive rats were determined by radioenzymatic assay. Significant decreases in the NE content of the mesenteric artery, renal artery and cardiac tissue from DOCA-Salt treated rats were found. In contrast the NE content of the inferior vena cava, abdominal aorta and mesenteric vein were not significantly decreased in the DOCA-Salt treated rats. There was no evidence of an altered disposition of DA and E in the vasculature of DOCA-Salt treated rats. The results of the study indicate that NE is selectively depleted in the cardiovascular system of this model of experimental hypertension and it is proposed that this property reflects different functional roles of individual elements of the vasculature in the DOCA-Salt hypertensive rat.