Contribution of the arterial tissue to vascular pathology in diabetes mellitus.

The contribution of peripheral arterial tissue to the development of vascular changes in Diabetes Mellitus (DM) was studied using the perfused tail artery from rats treated with Streptozotocin (25 mg/kg ip for 5 days). Dose-response curves to KCl and phenylephrine (PE) were constructed; the response to PE was significantly higher in DM than control arteries. No difference was found in the response to KCl between DM and controls. The response to vasodilators Acetylcholine (Ach), Papaverine, PAF-Acether were independent of the type of vasoconstrictor used to increase the vascular tone (KCl or PE). The response in the diabetic was significantly smaller than in the control arteries. The infusion of Indomethacin had no effect on the vasodilator response but enhanced the response to PE. The mechanical removal of endothelium increased the response to PE; presumably the vasoconstriction elicited by PE is counterbalanced by the release of vasodilators such as PGI2 and EDRF of endothelium origin. Infusion of Methylene Blue, that inhibits intracellular accumulation of c-GMP, in DM and control preparations partially blocked papaverine response and totally abolished the response to Ach and PAF-Acether. These data support an active participation of the peripheral arterial wall to the pathological changes present in diabetes mellitus.

Compartmental prostaglandin release by angiotensin II and arginine-vasopressin in rabbit isolated perfused kidneys.

The compartmental effects of angiotensin II (AII) and arginine-vasopressin (AVP) on renal prostaglandin (PG) formation were studied in the isolated perfused kidney of the rabbit by superfusion bioassay of venous and ureteral effluents (VE and UE) and radioimmunoassay (RIA). Comparable results were obtained with either bioassay or RIA when used to quantitate renal PG release. The effects on PG release into the VE were similar for AII and AVP, as were their pressor responses. However, their effects on PG release into the UE differed markedly. AII resulted in a 6-fold greater urinary efflux than venous of bioassayable PGs, whereas AVP-induced PG release into UE was slightly less than PG efflux into the VE at all doses of the peptide. The profile of released PGs varied according to the sampling source (VE or UE). Moreover, each peptide released a similar profile of PGs at all doses, i.e. UE PGE2 greater than PGF2 alpha greater than 6-keto PGF1 alpha; VE PGE2 greater than 6-keto PGF1 alpha greater than PGF2 alpha (TxB2 was not detected in either effluent). Thus, renal vascular PG release is similar for the vasoactive peptides, AII and AVP, whereas the urinary efflux of PGs is considerably greater in response to AII.

PGI2-specific antibodies administered in vivo suggest against a role for endogenous PGI2 as a circulating vasodepressor hormone in the normotensive and spontaneously hypertensive rat.

Immunoglobulins raised against 5,6-dihydro PGI2 crossreact with PGI2. When infused in vivo into the rat, these immunoglobulins are capable of 1) neutralising the vasodepressor effects (bolus or continuous infusion) of exogenous PGI2, 2) blocking the catabolism of exogenous 3H-PGI2 and prolonging its life-time in the circulation (t 1/2 approx 60 min) while that of 3H-PGE2 is unaffected, 3) trapping an endogenously produced substance which after extraction from blood and dissociation from the ligand-antibody complex, is immunoreactive with 6-keto PGF1 alpha-specific antiserum. Yet the anti-5,6-dihydro PGI2 immunoglobulins have no effect on resting arterial blood pressure both in the normotensive and spontaneously hypertensive rat. These experiments indicate that endogenously produced PGI2 does not play a significant systemic role in blood pressure control although in combination with other vasodilators it could still participate in the regulation of vascular tone at a local level.

Antibodies to 5,6-dihydroprostaglandin I2 trap endogenously produced prostaglandin I2 in the rat circulation.

Rabbit immunoglobulins raised against 5,6-dihydroprostaglandin I2 which crossreact with prostaglandin I2 were infused intravenously into Inactin-anaesthetised male adult rats. Clearance of intravenously administered [3H]prostaglandin I2 from the blood, which is normally rapid (t 1/2 approx. 45 s), was delayed strikingly in the presence of antibody (t 1/2 approx. 60 min). The antibodies also sequestered the endogeneously synthesized prostaglandin I2 and inhibited its metabolism. The rate of appearance of endogenous prostaglandin I2 in the circulation of the rat was measured in the following way: arterial blood samples (0.5 ml) were withdrawn before, during and at various time intervals (up to 180 min) after infusion of antibodies had terminated; the prostaglandins were extracted from the blood with ethanol, and the extracts were assayed by radioimmunoassay (before and after separation by high-pressure liquid chromatography) for the following prostaglandins: 6-keto-F1 alpha, E2, F2 alpha and 13,14-dihydro-15-keto-metabolites of E2 and F2 alpha. Rapid and specific time-related increments of prostaglandin I2 (detected serologically as 6-keto-F1 alpha) were observed. At 180 min these increases ranged from 1500- to 2500-fold over preinfusion levels. No significant increases were observed in the other prostaglandins measured; nor were there increases in 6-keto-F1 alpha when saline or immunoglobulins from non-immune plasma were infused into rats. When measured by these procedures, no appreciable differences in 6-keto-F1 alpha production were found between Japanese normotensive and spontaneously hypertensive rats.

Age-related differences in the urinary excretion of prostaglandin F2 alpha catabolites in the rat.

Tritium-labeled PGF2 alpha was administered i.v. into rats of varying ages (2, 4, 6 weeks and adult). Urine was collected and assayed for radioactive products by thin-layer-chromatography. Results showed a distinctly different urinary profile between the 2-week-old and the adult rat. While the urinary pattern from the 2-week-old rat gave a single less polar product than PGF2 alpha, the pattern from the adult rat gave products more polar than PGF2 alpha. Urine from the 4- and 6-week-old rats gave a mixture of these types of products. These results indicate that some prostaglandin catabolic pathway (likely the omega-oxidative system) is activated in vivo within the 4-6 week postnatal period in the rat.

Reduction in dietary vitamin E prevents onset of hypertension in developing spontaneously hypertensive rats.

Reduction in dietary vitamin E intake in developing spontaneously hypertensive rats abolished the onset of hypertension which is normally evident by 3 months of age.

Age-dependent increase in the formation of prostaglandin I2 by intact and homogenised aortae from the developing spontaneously hypertensive rat.

The prostaglandin I2 biosynthetic capacity of aortae from spontaneously hypertensive rats of various ages (1, 2, 3, 4, and 5 months) was investigated. An age-dependent increase in enzyme activity was observed reaching maximum by three months of age which correlated well with the age- related increase in the systolic blood pressure. These results support our notion that the enhanced aortic synthesis of the potent vasodepressor prostaglandin I2 by the spontaneously hypertensive rat likely represents an adaptive mechanism for the attenuation of the sustained elevation in blood pressure in this animal model.

Stability of authentic PGI2 during routine extraction. Clarification of the nature and sequence of products formed by the rat stomach including the origin of the ozonolysis products reported in 1971.

Authentic PGI2 and PGI2 formed by rat stomach homogenates were carried through a simple extraction and purification procedure to explore the feasibility of isolation of this biologically active bicyclic ether product of arachidonic acid. The integrity of PGI2 was followed throughout by bioassay on the rat blood pressure. In this system we recently reported that PGI2 has very potent hypotensive actions which are easily distinguishable from those observed for PGE2 (14). Our results indicate that PGI2 survives the initial extraction steps (i.e. ethanol extraction, diethyl ether - HCl extraction and methylation) up to the step involving thin layer chromatography with an acidic developing solvent system. This latter procedure converts PGI2 entirely into a stable derivative, 6-keto-PGF1alpha (3,8--10). Oxidative ozonolysis of the methyl ester acetate derivative of authentic 6-keto PGF1alpha reveals products identical to those reported by Pace-Asciak and Wolfe in 1971 (1) which are also produced from authentic PGI2. This data sheds new light into 1) the nature of the biological product formed by stomach homogenates, 2) its transformation into 6-keto PGF1alpha during purification and 3) the origin of the ozonolysis products in the experiments reported in 1971.

Enhanced formation of PGI2, a potent hypotensive substance, by aortic rings and homogenates of the spontaneously hypertensive rat.

Intact rings and homogenates of aorta from spontaneously hypertensive rats (SHR) contain enhanced capacity over normal rats (NR) to convert arachidonic acid into PGI2. The PGI2 synthetic system in SHR is stimulated to a greater extent than NR by norepinephrine. Indomethacin blocks this stimulation. PGE2 and PGF2alpha were detected in much smaller amounts in homogenates (undetected in rings) but their formation was not enhanced by the hypertensive tissue. The identity of PGI2 was based on 1) direct pharmacological assay on the rat blood pressure. In this system identical vasodepressor responses to PGI2 are observed after intracarotid and intrajugular administration 2) indirectly as 6-keto PGF1alpha isolated after incubation of aortic homogenates with tritiated arachidonic acid and 3) indirectly by GC-MS assay of PGE2, PGF2alpha and 6-keto PGF1alpha formed during incubation of aortic homogenates with excess unlabeled arachidonic acid. These results provide additional support to our recent hypothesis that PGI2, of aortic origin, might actively participate in the regulation of systemic blood pressure. Its enhanced formation by intact hypertensive vascular tissue reflects an increase in the number of enzyme molecules immediately available to the substrate. This could probably be an adaptive response to the elevated levels of catecholamines in the circulation.

Prostaglandin I2 has more potent hypotensive properties than prostaglandin E2 in the normal and spontaneously hypertensive rat.

The blood pressure lowering effects on PGI2 in the normal and spontaneously hypertensive rat are described. Comparison of dose response curves for PGI2 and PGE2 indicate that PGI2 is twice as potent as PGE2 in the normal rat and 3--4 times more active in the spontaneously hypertensive rat. Furthermore PGI2 is equiactive through intracarotid and intrajugular administration indicative of the complete lack of pulmonary inactivation. These findings supported by evidence of enhanced PGI2 synthesis in aorta during hypertension support the notion that PGI2 could participate in blood pressure control mechanisms.

Propranolol induces acute natriuresis by beta blockade and dopaminergic stimulation.

dl-Propranolol (0.8-1.6 mg/kg - h for 1 h) produced a transient two- to three-fold increase in sodium excretion in nondiuretic rats infused with Pitressin and aldosterone and in water diuretic rats. Sodium excretion increased more in rats depleted of renin by chronic Doca and salt administration than in rats maintained on a low salt diet. An angiotensin inhibitor (1,sarcosine-8,valine angiotensin II) decreased sodium excretion. Therefore the natriuresis was not mediated by antidiuretic hormone, aldosterone, or renin-angiotensin. d-Propranolol did not produce a natriuresis. Prior treatment with phenoxybenzamine did not prevent the natriuretic response but chlorisondamine pretreatment did. The natriuresis is produced by beta blockade and requires post ganglionic nerve function but is independent of alpha receptors. dl-Propranolol decreased heart rate and cardiac output but systemic pressure did not fall and renal blood flow increased. This suggests a dopamine-mediated renal vasodilation and natriuresis. Haloperidol and pimozide, both dopamine blocking agents with minimal beta blocking effects, prevented the natriuretic response. We conclude that propranolol may increase sodium excretion directly by blocking beta receptors in the distal nephron and indirectly by dopamine-mediated renal vasodilation.