Coronary arterial smooth muscle contraction by a substance released from platelets: evidence that it is thromboxane A2.

When human platelets are aggregated by thrombin, material is released that rapidly contracts strips of spirally cut porcine coronary artery. Prevention of the contraction by indomethacin suggested mediation by a prostaglandin. The contraction produced by aggregating platelets was unlike those produced by prostaglandins E2, F2alpha, G2, or H2, but resembled that evoked by thromboxane A2, which is formed by platelets during aggregation.

Indomethacin is a placental vasodilator in the dog. The effect of prostaglandin inhibition.

The effect of 8 mg/kg of indomethacin on uterine blood flow, prostaglandin production, and intraamniotic fluid pressure was examined in late pregnant dogs. Uterine blood flow was measured with 15 mum radiolabeled microspheres. Because we found that a significant percentage of the microspheres shunted through the placental circulation into the lungs, we calculated placental blood flow by adding the shunted microspheres through the placenta to the nonshunted microspheres in the placenta. Total uterine blood flow significantly increased from 271+/-69 ml/min during control period to 371+/-72 ml/min (P < 0.01) 30 min after indomethacin. This increase was attributable to the change in blood flow to the placental circulation (222+/-58 to 325+/-63 ml/min; P < 0.01). Associated with these hemodynamic changes we found an almost complete suppression of uterine prostaglandin E(2) production (1,654+/-305 to 51+/-25 pg/ml; P < 0.01) as measured by gas chromatography-mass spectrometry. In addition, we found that indomethacin treatment resulted in uterine relaxation as measured by intraamniotic fluid pressure changes (11.2+/-1.3 mm Hg to 8.5+/-1.2 mm Hg; P < 0.001). We conclude that indomethacin causes an increase in placental blood flow without any change in flow to the rest of the uterus, and that this dose of the drug inhibits greater than 95% of uterine prostaglandin production. In addition, indomethacin is responsible for uterine relaxation. The increase in placental blood flow after indomethacin is probably a result of uterine relaxation, which is secondary to prostaglandin synthesis inhibition.

Kinins: possible mediators of neonatal circulatory changes in man.

Bradykinin is a potent constrictor of the human umbilical artery and vein and the ductus arteriosus of the lamb in vitro at oxygen tensions above 40 mm Hg (comparable to those in the newborn infant). Bradykinin is also capable of producing remarkable dilatation of the pulmonary vasculature of the lamb. Theoretically, kinins are capable of effecting some of the rapid circulatory changes required of the neonate. The present study was undertaken to investigate the role of kinins as mediators of such changes. The concentration of bradykinin in the cord blood of 56 newborn infants at the time of birth was significantly higher than the blood level in adult subjects (12.8 +/- 4.3 ng/ml compared with 2.0 ng/ml or less). Cord arterial blood contained inactive kinin precursor (kininogen) and inactive kinin-releasing enzyme (kallikrein). Plasma kallikrein was activated, with subsequent kinin formation and kininogen depletion, by exposure to neonatal granulocytes or by a decrease in the temperature of cord blood from 37 to 27 degrees C. A comparable decrease in the temperature of umbilical arterial blood occurs at the time of birth. Activation of kallikrein by neonatal granulocytes was dependent on cell concentration and required oxygen tensions comparable to those in the neonate but above the range in the fetus. Granulocytes of the neonate, unlike those of adult subjects, lacked kininase activity.Thus, bradykinin can constrict and dilate vessels as required for the transition of fetal to neonatal circulation. Bradykinin can be produced in plasma of the newborn by decreases in temperature, such as occur in the umbilical blood at birth, and by exposure to granulocytes which are present in the circulation in increased numbers shortly after birth. We propose that bradykinin is produced at birth and may be a mediator of neonatal circulatory changes.

Increased clearance of antipyrine and d-propranolol after phenobarbital treatment in the monkey. Relative contributions of enzyme induction and increased hepatic blood flow.

The effects of phenobarbital treatment for 12 days on the regional distribution of blood flow and on the disposition of two model drugs, antipyrine and d-propranolol, have been determined in six unanesthetized rhesus monkeys. Phenobarbital significantly increased total hepatic blood flow from 179+/-15 to 239+/-27 ml/min. Liver weight was increased to a similar degree (34%) in phenobarbital-treated animals as compared to control monkeys. The clearance of both antipyrine and d-propranolol was increased and the half-life decreased significantly by phenobarbital. Analysis of the data by a perfusion-limited pharmacokinetic model showed that the changes in antipyrine clearance were due almost entirely to enzyme induction. On the other hand, with d-propranolol, the increase in liver blood flow contributed as much to the enhanced clearance as did the stimulation of drug metabolism. The mechanism by which phenobarbital produces the frequently observed increase in drug clearance, therefore, depends upon the initial clearance value of the drug. For low clearance drugs like antipyrine, clearance changes occur largely as a result of enzyme induction. With higher clearance drugs, the effects of increased hepatic blood flow become progressively more important the greater the initial clearance value.

Prostacyclin produced by the pregnant uterus in the dog may act as a circulating vasodepressor substance.

Uterine production of PGI2 (prostacyclin) was quantitated in late pregnant dogs to evaluate if PGI2 could act as circulating vasodepressor substance in pregnancy. In five anesthetized, laparotomized dogs, the uterine venous plasma concentration of 6-keto PGF1 alpha (the in vitro hydrolysis product of PGI2) was 6.7 +/- 1.9 ng/ml and the arterial plasma concentration was 2.6 +/0 0.8 ng/ml. In four nonpregnant female dogs the arterial plasma concentration of 6-keto PGF1 alpha was consistently below 0.2 ng/ml. In eight pregnant dogs we also evaluated the ability of the pregnant uterus to inactivate PGI2 by comparing the hypotensive response to increasing doses of PGI2 infused into the uterine artery to the hypotensive response to increasing doses of PGI2 infused into the inferior vena cava. In addition, the effect of PGI2 infused into the uterine artery on uterine blood flow and intraamniotic fluid pressure was evaluated. The dose-response curves of intrauterine and intravenous PGI2 in causing systemic hypotension were identical suggesting that the pregnant uterus does not inactivate infused PGI2. Intrauterine PGI2 had no consistent effect on uterine hemodynamics although it did increase intraamniotic fluid pressure significantly. These data demonstrate that the pregnant uterus has the capacity to produce large quantities of PGI2 which is not inactivated in the uterus and therefore can appear in the arterial blood to exert a systemic vasodepressor effect.

Elevation of beta-adrenergic receptor density in human lymphocytes after propranolol administration.

Abrupt withdrawal after the chronic administration of propranolol has resulted in clinical syndromes that suggest adrenergic hypersensitivity. The effect of propranolol administration and withdrawal on beta-adrenergic receptors was studied in human lymphocyte membranes. Receptor density was quantitated by direct binding assays with the radioligand [125I]iodohydroxybenzylpindolol. Administration of propranolol (160 mg/d) for 8 d resulted in trough plasma levels of approximately 35 ng/ml. By day 5 of propranolol administration the density of beta-adrenergic receptors had increased 43 +/- 4% (P less than 0.01) above pretreatment levels. Abrupt withdrawal of propranolol was followed by the disappearance of propranolol from the plasma within 24 h. The density of beta-adrenergic receptors did not return to pretreatment level for several days. Physiologic supersensitivity of beta-adrenergic receptor-mediated responses was suggested by the appearance of significant increases in the orthostatic change in heart rate (P less than 0.05) and the orthostatic change in the heart rate-systolic blood pressure product (P less than 0.01) during the first 48 h after propranolol withdrawal. These data show that propranolol administration leads to an increase in the density of beta-adrenergic receptors in human tissue. The results are consistent with the hypothesis that some of the untoward effects observed after abrupt discontinuation of propranolol are caused by beta-receptor-mediated adrenergic hypersensitivity.

Urinary prostaglandins. Identification and origin.

Human urine was analyzed by mass spectrometry for the presence of prostaglandins. Prostaglandin E2 and F2alpha were detected in urine from females by selected ion monitoring of the prostaglandin E2-methylester-methoxime bis-acetate and the prostaglandin F2alpha-methyl ester-Tris-trimethylsilylether derivative. Additional evidence for the presence of prostaglandin F2alpha was obtained by isolating from female urine an amount of this prostaglandin sufficient to yield a complete mass spectrum. The methods utilized permitted quantitative analysis. The origin of urinary prostaglandin was determined by stimulating renal prostaglandin synthesis by arachidonic acid or angiotensin infusion. Arachidonic acid, the precursor of prostaglandin E2, when infused into one renal artery of a dog led to a significant increase in the excretion rate of this prostaglandin. Similarly, infusion of angiotensin II amide led to a significantly increased ipsilateral excretion rate of prostaglandin E2 and F2a in spite of a simultaneous decrease in the creatinine clearance. In man, i.v. infusion of angiotensin also led to an increased urinary eliminiation of prostaglandin E. These results show that urinary prostaglandins may originate from the kidney, indicating that renally synthesized prostaglandins diffuse or are excreted into the tubule. Thus, urinary prostaglandins are a reflection of renal prostaglandin synthesis and have potential as a tool to delineate renal prostaglandin physiology and pathology.

Synthesis of prostaglandins by the rat renal papilla in vitro. Mechanism of stimulation by angiotensin II.

1. The biosynthesis of prostaglandins in the rat renal papilla was studied in a whole-cell preparation in vitro. Prostaglandins recovered from the incubation medium were identified by gas chromatography-mass spectrometry as prostaglandin E2 and prostaglandin F2alpha. Quantitative estimates of prostaglandin output were obtained by bioassay and confirmed by selected ion monitoring. 2. Prostaglandin biosynthesis was enhanced by exogenous arachidonic acid and also by triglyceride lipase, indicating that arachidonic acid released from papillary triglycerides is readily available for prostaglandin biosynthesis. 3. Angiotensin II (10--100 ng/ml) stimulated the biosynthesis of both prostaglandin E2 and prostaglandin F2alpha, thus increasing prostaglandin levels in both the incubation medium and the tissues. 4. The mechanism whereby angiotensin II stimulates prostaglandin biosynthesis was investigated using the isotope dilution technique. In the presence of [14-C]-arachidonic acid, angiotensin II stimulated the output of more prostaglandin that had a significantly lower specific activity than the controls. Angiotensin II therefore increased the availability of endogenous, non-labelled substrate for prostaglandin biosynthesis. This conclusion was supported by experiments in which enough arachidonic acid was added to make the kinetics of prostaglandin synthesis zero order. Under such conditions angiotensin II failed to cause any further increase in prostaglandin synthesis. 5. It is concluded that angiotensin II controls prostaglandin biosynthesis in the renal papilla by regulating the availability of free precursor. Possible mechanisms for increased levels of free arachidonic acid could be the activation of a tissue acyl hydrolase or decreased utilization of fatty acids.

Simultaneous measurement of cardiac output and its distribution with microspheres in the rat.

The radioactive microsphere method was used to estimate simultaneoulsy the cardiac output and its distribution in the same rat by the use of an arterial reference sample obtained during microsphere distribution. Sufficient microspheres were injected so that all counted samples contained more than 400 spheres. No haemodynamic changes occurred during microsphere injection. The results for cardiac output of 253 +/- 11 ml/min per kg body weight agree with published estimates utilizing other techniques. The distribution of cardiac output also agrees with most published reports. This method should allow the rat to be conveniently used for certain haemodynamic studies when cardiac output and organ blood flow are necessary.

Hemodynamic effects of platelet activating factor in the dog kidney in vivo.

The effect of platelet activating factor (PAF) on renal hemodynamics and function was examined in anesthetized dogs. The infusion of PAF into the renal artery at 5, 10, and 20 ng X min-1 X kg-1 body weight resulted in dose-dependent reductions in renal blood flow, glomerular filtration rate, urine volume, and urinary sodium excretion, whereas the infusion of vehicle alone in the contralateral kidney did not result in significant changes in these parameters. The maximum decrease expressed as the percent change from baseline was 22.2 +/- 1.7% for renal blood flow, 50.8 +/- 11% for glomerular filtration rate, 67.3 +/- 4.2% for urine volume, and 69.0 +/- 8.5% for urinary sodium excretion, respectively. These renal effects were not accompanied by significant alterations in systemic arterial blood pressure and heart rate. Pretreatment with indomethacin to block prostaglandin synthesis enhanced the effect of PAF on kidney function. Our data demonstrate that, unlike the rat kidney, intrarenal PAF infusion into the intact dog results in vasoconstriction and serve reduction in glomerular filtration rate.

Propranolol increases prostacyclin synthesis in patients with essential hypertension.

We tested the hypothesis that vascular prostacyclin synthesis is increased by propranolol and could account for some of the drug's antihypertensive effect. We studied 10 white patients with mild essential hypertension in a randomized, double-blind design to assess the effects of indomethacin with or without the addition of propranolol on blood pressure and vascular prostacyclin biosynthesis, as assessed by the urinary excretion of the major enzymatically produced metabolite of prostacyclin, 2,3-dinor-6-keto-prostaglandin F1 alpha (PGF1 alpha), F1 alpha (PGF1 alpha), measured by gas chromatography-mass spectrometry. Seven patients responded to propranolol with a lowering of mean arterial blood pressure in both supine and upright postures. The fall in mean arterial blood pressure (-14.1 +/- 2.1 mm Hg sitting; -17.4 +/- 1.7 mm Hg supine) with propranolol alone was significantly greater than that produced when propranolol was given to patients receiving indomethacin (-7.8 +/- 1.9 mm Hg sitting; -7.7 +/- 3.0 mm Hg supine). Our drug-responsive patients demonstrated a significantly lower excretion rate of 2,3-dinor-6-keto-PGF1 alpha than was found in an age and sex-matched group of normal volunteers. With propranolol treatment, drug-responsive patients showed a significant increase in the excretion of 2,3-dinor-6-keto-PGF1 alpha, such that the mean excretion was not significantly different from that in normal volunteers. Indomethacin caused a significant rise in mean arterial blood pressure and a significant fall in 2,3-dinor-6-keto-PGF1 alpha excretion, and it blocked the rise in urinary 2,3-dinor-6-keto-PGF1 alpha associated with propranolol therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in the responses of the sympathetic nervous system and renin in borderline hypertension.

We investigated the effect of stimuli activate the sympathetic nervous system on plasma catecholamines, renin activity, urinary metanephrine and normetanephrine, and various hemodynamic parameters in normal subjects (NIs) and borderline hypertensive (BH) subjects. No differences were observed in sympathetic nervous system activity or renin activity when the subjects were in the resting state on a 150 mEq sodium diet. However, the BH group exhibited greater responses in terms of plasma catecholamines and plasma renin activity in response to sodium deprivation and treadmill exercise. Although hemodynamic differences in the cold pressor test and handgrip exercise did not emerge, the radio of atrial size decrement to venous tone increment during the Valsalva maneuver was significantly reduced in the BH group. The investigations suggest that in the basal state, BH subjects have appropriate levels of activation of the sympathetic and renin systems for a normal level of pressure but that perturbations of pressure and volume factors lead to unmasking of abnormalities in regulation of both systems. The data are also consistent with the suggestion that venous compliance is reduced in these patients.

Safety of single large oral doses of guanethidine.

The cardiovascular effects of single 100-mg doses of guanethidine were assessed in hypertensive patients by measurement of arterial pressure, heart rate, and systolic time intervals. In the 3-hr period following a dose there was no evidence of an inotropic effect. Therefore, large doses of guanethidine as required for the guanethidine loading regimen would seem to be safe even in patients in whom inotropic effects of released catecholamines would be contraindicated.

Beta-adrenergic receptors in the elderly are not less sensitive to timolol.

The elderly have been reported to be less sensitive to the beta-adrenergic blocking effect of propranolol. However, propranolol is a racemate, and age-related changes in stereoselective metabolism or protein binding could confound interpretation of the data. To avoid these problems, we studied timolol in 12 young and 12 elderly healthy subjects. The dose of isoproterenol required for a heart rate increase of 25 bpm (I25) was determined before and 2 hours after an oral 10 mg dose of timolol. A dose ratio (DR) was calculated for each subject as the I25 after timolol/I25 before timolol. The binding constant for timolol binding to the receptor was calculated as the plasma timolol concentration divided by (DR-1). The I25 for the elderly group was significantly greater than the I25 for the young group, but the timolol binding constant was the same for both groups. We conclude that, although the elderly are less sensitive to isoproterenol, they are not less sensitive to timolol, and thus our data do not implicate a change in the interaction of beta-adrenoceptors with antagonists.

Platelet alpha 2-adrenergic receptor responsiveness is increased in elderly men but not in elderly women.

We ascertained platelet alpha 2-adrenergic receptor responsiveness in healthy young and elderly men and women by determining the minimum concentration of methylnorepinephrine, a selective alpha 2-adrenergic receptor agonist, required to initiate the primary and secondary aggregation response in platelet-rich plasma. We observed that platelets from elderly men required a smaller concentration of methylnorepinephrine to stimulate primary aggregation than did platelets obtained from young men. However, the concentration of alpha 2-adrenergic receptor agonist to trigger the secondary aggregation response did not vary with age. There was no difference in the responsiveness of platelets from young and elderly women for either the primary or secondary aggregation response. We conclude that platelet alpha 2-adrenergic receptor responsiveness is increased with age rather than decreased, as predicted from some studies of the density of platelet alpha 2-adrenergic receptors and their coupling to adenylyl cyclase. Furthermore, the increase in alpha 2-adrenergic receptor responsiveness is gender specific.

The relationship of plasma guanethidine levels to adrenergic blockade.

Seventeen hypertensive patients receiving guanethidine for therapy were studied to determine the relationship of guanethidine plasma levels to adrenergic blockade. Plasma levels of guanethidine were measured by gas chromatography-mass spectrometry, and adrenergic blockade was defined by determining the venous reflex response to Valsalva maneuver or deep breath. A significant correlation was found between the change in the venous reflex response and the fall in mean standing pressure when guanethidine is given to patients maintained on a sodium restricted diet. A linear relationship was found between dose and plasma guanethidine concentration (p less than 0.0001), but there was a 6-fold interindividual variation in the plasma levels resulting from any given dose. Adrenergic blockade occurred when plasma levels were 8 ng/ml or higher. These results indicate that the large individual variation in dose requirements for the hypotensive effects of guanethidine most likely is not due to requirements for greatly different plasma levels of the drug; that the variation must result from pharmacokinetic determinants of differing plasma levels between individuals or from other factors, such as increased plasma volume, which maintain elevated arterial pressure in the face of adrenergic blockade.

The balance between vascular alpha- and beta-adrenoceptors is not changed in the elderly.

It has been suggested that beta-adrenoceptor-mediated functions are diminished with aging and that these responses are reduced to a greater extent than are alpha-adrenoceptor-mediated responses. The resulting imbalance in the elderly may produce an increased vascular resistance from the unopposed alpha-adrenoceptor stimulation in the peripheral vasculature. To evaluate this hypothesis, we studied 12 healthy elderly and 12 healthy young subjects during a graded infusion of epinephrine and compared blood pressure response, vascular resistance, and calf blood flow as determined by venous occlusion plethysmography. In both groups, heart rate increased, blood flow to the leg increased, and vascular resistance fell in response to epinephrine infusion, but in the elderly the systolic blood pressure failed to rise as it did in the young subjects. From these data we conclude that the overall vascular response to epinephrine does not change with age and that the balance between beta-adrenoceptor-mediated vasodilation and alpha-adrenoceptor-mediated vasoconstriction is therefore unchanged in the elderly.

The effect of age on the sensitivity of the alpha 1-adrenoceptor to phenylephrine and prazosin.

Certain beta-adrenoceptor-mediated functions seem to diminish with age; however, information on alpha-adrenoceptor-mediated function is sparse and often conflicting but overall suggests little age-related change. To assess an age-related alteration in the responsiveness to an alpha 1-adrenergic agonist and to estimate changes in the apparent affinity of the alpha 1-adrenoceptor for the antagonist prazosin, we infused phenylephrine into 12 healthy elderly subjects and 12 healthy young subjects before and after an oral dose of prazosin, and we compared the shift in the dose-response curves for the two groups. With this protocol we were unable to detect any age-related decline in sensitivity of the alpha-adrenoceptor to either agonist or antagonist. However, oral prazosin resulted in higher plasma concentrations and a consistently greater hypotensive effect in the elderly subjects than in the young subjects. We concluded that there was no difference in alpha 1-adrenoceptor sensitivity in the elderly persons, but that the kinetics of prazosin may be altered and that the response of the blood pressure to prazosin was increased because of the kinetic changes and possibly the physiologic changes associated with aging.

The antihypertensive efficacy of hydrochlorothiazide is not prostacyclin dependent.

We tested the hypothesis that vascular prostacyclin synthesis is stimulated by hydrochlorothiazide and could account for some of the drug's antihypertensive effect. We studied 13 patients with mild essential hypertension in a randomized, double-blind design to assess the effects of indomethacin on hydrochlorothiazide's ability to lower blood pressure, alter body weight, stimulate plasma renin activity, and modulate vascular prostacyclin biosynthesis as assessed by the urinary excretion of the major enzymatically produced metabolite of prostacyclin, 2,3-dinor-6-keto-prostaglandin F1 alpha (PGF1 alpha), measured by GC/MS. Administration of hydrochlorothiazide, 50 mg daily for 2 weeks, was associated with a significant decrease in both systolic and diastolic blood pressure in both supine (systolic, 148 +/- 3 to 136 +/- 3 mm Hg; diastolic, 97 +/- 2 to 94 +/- 3 mm Hg) and upright (systolic, 151 +/- 4 to 131 +/- 2 mm Hg; diastolic, 103 +/- 2 to 97 +/- 3 mm Hg) positions. Hydrochlorothiazide administration resulted in a 1 kg weight loss and stimulation of plasma renin activity from 1.7 +/- 0.4 to 5.3 +/- 1.1 ng angiotensin I/ml/hr. However, the urinary excretion of 2,3-dinor-6-keto-PGF1 alpha was unchanged after administration of hydrochlorothiazide (86 +/- 13/ng/gm creatinine during placebo, 74 +/- 13 ng/gm during week 1 of hydrochlorothiazide, and 70 +/- 9 ng/gm during week 2 of the drug). Administration of indomethacin, 50 mg twice a day, resulted in greater than 60% inhibition of 2,3-dinor-6-keto-PGF1 alpha excretion but did not affect the antihypertensive response to hydrochlorothiazide. Indomethacin did not oppose the diuretic effect of hydrochlorothiazide as assessed by weight loss but did attenuate the rise in plasma renin activity. Our data demonstrate that the blood pressure-lowering effect of a thiazide diuretic does not require enhanced prostacyclin synthesis and the cyclooxygenase inhibitor indomethacin does not antagonize the antihypertensive efficacy of hydrochlorothiazide.

Lack of an effect of age on the response to clonidine.

The plasma concentration and appearance rate of norepinephrine are increased in the elderly. A hypothesis to explain this observation is that the elderly have a diminished response of the alpha 2-adrenoreceptor in the brainstem that modulates peripheral sympathetic tone. To evaluate the effect of age on alpha 2-adrenoreceptor function, we studied 12 healthy elderly subjects and 12 healthy young volunteers and compared the decrease in plasma norepinephrine and blood pressure in response to increasing doses of orally administered clonidine. We found that, for the same plasma clonidine concentration, the blood pressure and plasma norepinephrine concentration fell equivalently in both groups. These data imply that the increased plasma norepinephrine and the elevated blood pressure in the healthy elderly population do not appear to be secondary to a decrease in alpha 2-adrenergic response to an agonist in the central nervous system.