Adenosine A(1) receptors and vascular reactivity.

AIM: Blood pressure is higher in A(1) receptor knock-out (A(1)R-/-) mice than in wild type litter mates (A(1)R+/+) and we have examined if this could be related to altered vascular functions. METHODS: Contraction of aortic rings and mesenteric arteries were examined. To examine if the adenosine A(1) receptor-mediated contraction of aortic muscle was functionally important we examined pulse pressure (PP) and augmentation index (AIX) using a sensor that allows measurements of rapid pressure transients. RESULTS: Contraction of aortic rings to phenylephrine and relaxation to acetylcholine were similar between genotypes. The non-selective adenosine receptor agonist N-ethyl carboxamido adenosine (NECA) enhanced the contractile response, and this was eliminated in aortas from A(1)R-/- mice. However, in mesenteric arteries no contractile response was seen and adenosine-mediated relaxation was identical between studied genotypes. A(2B) adenosine receptors, rather than A(2A) receptors, may be mainly responsible for the vasorelaxation induced by adenosine analogues in the examined mouse vessels. PP was higher in A(1)R-/- mice, but variability was unaltered. AIX was not different between genotypes, but the NECA-induced fall was larger in A(1)R-/- mice. CONCLUSIONS: The role of adenosine A(1) receptors in regulating vessel tone differs between blood vessels. Furthermore, contractile effects on isolated vessels cannot explain the blood pressure in A(1) knock-out mice. The A(1) receptor modulation of blood pressure is therefore mainly related to extravascular factors.

Perinatal development influences mechanisms of bradykinin-induced relaxations in pulmonary resistance and conduit arteries differently.

OBJECTIVE: As bradykinin (BYK) relaxes conduit (EPA) and resistance (RPA) pulmonary arteries from both perinatal and adult lungs, we investigated whether this vasodilator's relaxation-mechanisms were altered during perinatal development, differed between EPA and RPA and differed with other endothelium-dependent vasodilators, acetyicholine (ACH) and substance P (SP). METHODS: Arteries from mature foetal (5 days), neonatal (approximately 5 min), newborn (60-84 h) and adult pigs (> or =6 months) were isolated, mounted for in vitro isometric force recording, activated with PGF(2alpha) (30 micromol/l) and relaxed with BYK (10 pmol/l-1 micromol/l), SP (10 pmol/l-0.1 micromol/l) or ACH (1 nmol/l-1 mmol/l). RESULTS: (i) BYK: L-NAME (100 micromol/l) attenuated relaxations in foetal EPA ( approximately 55%) but nearly abolished them in the adult ( approximately 80%). In RPA, L-NAME nearly abolished ( approximately 90%) relaxations in the foetus and this effect diminished progressively with age to approximately 20% in the adult. Indomethacin (IND, micromol/l) attenuated relaxations in neonatal (approximately 25%), new-born and adult EPA (both approximately 45%). Together, L-NAME and IND abolished relaxations in all EPA and in neonatal RPA but not in older RPA. SKF525a (100 micromol/l) attenuated relaxations in foetal RPA ( approximately 4%), diminishing in the adult RPA to approximately 10%. Together, SKF52Sa and L-NAME largely abolished relaxations in postnatal RPA (approximately 80%). Activation with K(+)=125 mmol/l attenuated relaxations in adult EPA (approximately 80%), foetal RPA ( approximately 45%) and neonatal RPA (approximately 75%) and abolished relaxations in RPA from older ages. (ii) ACH: L-NAME abolished relaxations in new-born EPA and RPA. In adult EPA, combined L-NAME and IND moderately attenuated relaxations. (iii) SP: Combined application of L-NAME and IND attenuated relaxations to a similar degree in new-born and adult EPA and RPA. CONCLUSIONS: In postnatal EPA, BYK-relaxations depend completely on prostaglandin- and NO-synthesis whereas those to SP (at all ages) and ACH (in the adult) do not. In RPA, BYK-relaxations develop from being completely dependant on the sole release of NO (foetus) to being almost completely independent of it (adult), a situation mimicked partially by SP but not by ACH, which, in new-born RPA is completely dependent on NO. BYK-relaxations in postnatal RPA depend on the release of a hyperpolarising factor generated through an SKF525a-sensitive pathway in conjunction with NO. The mechanisms of endothelium-dependent BYK-relaxations in the pulmonary vascular bed undergo diverging alterations, depending on the stage of development and arterial size/function. These changes are specific for BYK as they differ from those obtained from ACH or SP.

Up-regulation of bradykinin response in rat and human bladder smooth muscle.

PURPOSE: Responses to bradykinin were investigated in vitro in isolated control and hypertrophic smooth muscle strips from rat bladder. MATERIALS AND METHODS: Bladder hypertrophy was induced by a 10-day period of partial urinary outflow obstruction. In addition, human bladder strips were also investigated. RESULTS: Bradykinin (1 nM. to 1 microM.) caused contractions in all tissues studied. In the freshly isolated rat bladder preparations bradykinin induced contractions were similar and of small amplitude in control and hypertrophic tissues. After a 4-hour equilibratory period contractile responses to bradykinin and the B1 specific bradykinin receptor agonist desArg9 bradykinin were slightly increased in the controls but there was approximately a 6-fold increase in the hypertrophic muscle strips. After 4 hours of equilibration the human bladder strips showed a smaller but still significant increase in contractile response to bradykinin. Indomethacin, a cyclooxygenase inhibitor, almost abolished the increased response, which suggests that prostanoids are involved in the up-regulated response. The protein synthesis inhibitor cycloheximide inhibited up-regulation by approximately 50% in hypertrophic and control muscle strips from rat bladder and normal muscle from human bladder. CONCLUSIONS: These results demonstrate that bradykinin receptor responses are present in rat and human detrusor muscle and they can be up-regulated in vitro. Experiments on hypertrophic rat bladder revealed that this process is enhanced in hypertrophy.

Changes in ANP responsiveness of normal and hypertensive porcine intrapulmonary arteries during maturation.

Pulmonary vascular resistance falls rapidly after birth, but endothelium-dependent relaxation is relatively poor during the perinatal period. Atrial natriuretic peptide (ANP) is a potent vasodilator; however, its role in the process of perinatal adaptation is uncertain. Porcine intrapulmonary conduit arteries (IPA) from fetal, newborn (< 5 min), 3-, 6-, and 17-d-old, and adult pigs, and from piglets made hypoxic from 0 to 3, 3 to 6, or 14 to 17 d, were isolated and mounted for isometric force recording. Rings were precontracted with prostaglandin-F2 alpha (PGF2 alpha, 10 microM) or KCl (40 mM). ANP was added cumulatively (10 pM to 100 nM). C-type natriuretic peptide (CNP) was added as a single concentration of 100 nM. Accumulation of cGMP under basal conditions and stimulated by ANP or CNP was measured by radioimmunoassay system. Frozen sections of lung tissue were incubated with 125I-labeled alpha-ANP, and binding site density was assessed on IPA with an image analysis system. ANP relaxed IPA in pigs at all ages, but the effect was significantly greater at 6 and 17 d of age. Hypoxia in animals from 14 to 17 d old impaired ANP-induced relaxation. CNP relaxed IPA poorly: < 12% at all ages. ANP increased cGMP accumulation in both normal and hypoxic animals. CNP did not increase cGMP generation in IPA from normal animals but did so in IPA from 3-d-old hypoxic animals. ANP-specific binding sites were demonstrated on the pulmonary artery smooth muscle cells, with greater binding in the young animals. The increased relaxant responses to ANP during adaptation may be important in maintaining low pulmonary vascular resistance. In contrast, CNP was largely ineffective in relaxing pulmonary arteries.

Maturation of the response to bradykinin in resistance and conduit pulmonary arteries.

OBJECTIVE: Immaturity of the endothelial-dependent relaxation is thought to be characteristic of the newborn pulmonary elastic arteries. In adulthood, the reactivity of different pulmonary arterial segments varies. Therefore, we investigated the presence of endothelial heterogeneity in perinatal porcine pulmonary arteries and compared it with the adult by studying the bradykinin-, substance P- and acetylcholine-induced relaxations in different arteries. METHODS: Three types of pulmonary arteries (large conduit elastic, distal branching and resistance-sized; in situ diameters 0.7-1.7, 0.3-0.5 and 0.1-0.2 mm, respectively) were isolated from lungs of adult (nine months), young (60-84 h), newborn (4 min) and near-term foetal pigs. They were mounted for isometric force recording, contracted first with K+ = 125 mmol/l (reference contraction). Cumulative concentration-response curves to acetylcholine, substance P or bradykinin were obtained from prostaglandin F2 alpha (30 mumol/l) precontracted vessels. The effects of captopril and O2(95 or 8%) were also determined. Experiments were terminated by adding 100 mumol/l papaverine, obtaining maximal relaxation, which was used for normalising relaxations. RESULTS: (i) Acetylcholine: In resistance arteries, relaxations were absent in the newborn and the adult. In conduit arteries, they were present from 60-84 h onward. (ii) Substance P: In resistance arteries, relaxations were only present in the adult. In the other two types of arteries, rudimentary relaxations were present from the mature foetal stage onward. (iii) Bradykinin: In resistance arteries, identical relaxations were present at all ages which, in the foetus and the adult, were insensitive to changes in O2 levels (95 to 8%). In conduit arteries, concentration-dependent relaxations were present from birth, increasing in amplitude with age and these were potentiated by captopril. Foetal conduit arteries relaxed to the single application of 0.1 mumol/l bradykinin, indicating age-dependent tachyphylaxis. CONCLUSIONS: (i) Bradykinin is unique among endothelium-dependent vasodilators in being able to relax all vascular segments, at all ages, subject to tachyphylaxis and bradykinin-breakdown but independent of the prevailing O2 concentration. (ii) Heterogeneity of the relaxations between conduit and resistance arteries is evident from the mature foetal stage onward. (iii) The type of agonist, the type of vessel and the age each independently determine the presence or absence of endothelial relaxations. Therefore, the perinatal pulmonary circulation is not immature with respect to endothelial-dependent relaxation; rather, the nature of this process changes within the perinatal period and between birth and adulthood.

Developmental changes in binding sites and reactivity for CGRP and VIP in porcine pulmonary arteries.

During postnatal adaptation pulmonary arteries dilate. CGRP and VIP are pulmonary vasodilators. In this report, porcine lungs from newborn to adult were studied. Radiolabeled ligand binding and autoradiography showed CGRP binding sites on the endothelium of pulmonary arteries and veins, which increased postnatally, and VIP binding sites on smooth muscle, which decreased. Isolated conduit arteries relaxed normally (initially endothelium dependent) in response to CGRP from birth. VIP first caused relaxation at 10 days and was endothelium dependent. Age-related changes in receptor binding density were not always reflected in an appropriate alteration in pharmacological response.

ATP-dependent K+ channel activation in isolated normal and hypertensive newborn and adult porcine pulmonary vessels.

The role of an ATP-dependent K+ channel (K(ATP)+) relaxation in the porcine pulmonary vasculature from birth to adulthood was investigated in vitro using levcromakalim on isolated, prostaglandin F2alpha (30 microM)-precontracted conduit arteries (CA), resistance arteries (RA), and veins (PV). Vessels from neonatal pulmonary hypertensive piglets exposed to chronic hypobaric hypoxia (CHH, 51 kPa) for 3 d, either from birth or from 3 d of age were also studied. Levcromakalim relaxed all vessels in a concentration- and glibenclamide-sensitive manner. In normal CA, the maximal extent of relaxation and sensitivity (EC50) increased between birth and 17 d. Endothelium-removal increased EC50 at 17 d only. Indomethacin (10 microM), but not N(G)-monomethyl-L-arginine (L-NMMA) (30 microM), inhibited relaxation in CA from newborn, 3-d-old, and adult animals. In RA, levcromakalim-induced relaxations did not change during development and endothelium-removal attenuated relaxations in 3-d-old but not in adult animals. At both ages in RA, L-NMMA attenuated relaxations and subsequent addition of L-arginine (1 mM) restored them. In PV, maximal relaxation increased between birth and 6 d with no change of EC50. At all ages, relaxation was partially endothelium-dependent and inhibited by L-NMMA (except in the newborn). Indomethacin only attenuated relaxations in veins from 6- and 17-d-old animals. CHH did not influence relaxant responses in CA and PV but decreased EC50 in RA. Thus K(ATP)+ channel activation caused relaxation from birth onward in all vascular segments with varying endothelium dependence. CHH did not affect relaxation in the large vessels and up-regulated those in RA. These findings indicate a possible role for K(ATP)+ channels during normal adaptation and a potential therapeutic role in the management of pulmonary hypertensive newborn infants.

Chronic hypoxia inhibits postnatal maturation of porcine intrapulmonary artery relaxation.

Neonatal pulmonary hypertension is associated with increased pulmonary vascular reactivity. We studied the responses of isolated porcine intrapulmonary arteries after exposure of piglets to chronic hypobaric hypoxia (CHH) from 0 to 2.5, 3 to 6, or 14 to 17 days of age. CHH inhibited the postnatal development of endothelium-dependent vasorelaxation to acetylcholine (ACh) and the calcium ionophore A-23187. Basal accumulation of guanosine 3', 5'-cyclic monophosphate (cGMP) was unaffected, but cGMP response to ACh was inhibited. Endothelium-independent relaxation to nitric oxide (NO) and zaprinast (a phosphodiesterase inhibitor) was also inhibited, but cGMP accumulation in response to these agonists was normal. The ability of sodium nitroprusside (SNP) to cause vasorelaxation and increase cGMP accumulation was unaffected. Contractile responses to potassium chloride and prostaglandin F2 alpha (PGF2 alpha) were similar to normal after exposure from birth and 3 days and were decreased in the older group, but the ability of NG-monomethyl-L-arginine acetate to increase PGF2 alpha-induced contractions decreased. Thus exposure of newborn piglets to CHH causes 1) no increase in contractile responses and 2) impairment of endothelium-dependent and -independent relaxation by impairing signal transduction mechanisms involved in the release of NO and the effectiveness of cGMP.

Growth resistance-sized arteries in response to bladder hypertrophy in the rat: time-course, DNA-synthesis and LDH-isoform pattern.

Bladder growth was induced by partial urethral obstruction. Bladder hypertrophy was evident at 53 h after obstruction and continued over a 6 weeks period. Small bladder arteries were taken from fixed anatomical locations of the bladder circulation, mounted in a small vessel myograph and the optimal diameter for maximal isometric force development was determined (Lmax K+ = 125 mM stimulation). Bladder hypertrophy was associated with an enlarged Lmax from 53h onward (compared with sham-operated controls) and Lmax continued to increase until 10 days after urethral obstruction. Between 10 days and 6 weeks no further increase of the diameter was observed. Increased diameters in vitro were accompanied by a transiently increased [3H] Thymidine uptake in the small arteries which peaked at 53 h after obstruction but was still above background at 10 days. At this time point, small arterial growth was associated with a significant relative increase in the M isoform of LDH as determined with agarose electrophoresis on tissue homogenates. Thus organ growth induced small vessel growth in the rat is characterized by a rapid onset, increased but transient DNA-turnover and LDH-isoform changes. The latter mimic changes seen in other types of smooth muscle growth.

Structure and mechanics of growing arterial microvessels from hypertrophied urinary bladder in the rat.

Rat bladder hypertrophy, induced by a partial ligation of the urethra, was used to study the accompanying changes of microvascular smooth muscle mechanics, pharmacology and morphology. A segment of a microarterial vessel to the bladder was taken from a defined anatomical location and studied in a wire myograph in vitro at the length for maximal isometric force development (Lmax). After 10 days of ligation, bladder hypertrophy resulted in a microvascular growth response compared to non-operated controls which was characterized by (i) an increase of the calculated diameter at Lmax from 134 +/- 5 microns to 222 +/- 19 microns; (ii) an increase of the media thickness from 22.4 +/- 1.9 microns to 32.2 +2- 3.0 microns; (iii) an increase of the active tension from 1.42 +/- 0.28 mN/mm to 3.06 +/- 0.33 mN/mm; (iv) no change of the wall/lumen ratio (from 0.83 +/- 0.10 to 0.79 +/- 0.15). Normalized length/force relations (active, passive and total) did not differ significantly between microarteries from control and hypertrophic bladders. Microvascular smooth muscle growth was also associated with a decreased sensitivity to K(+)-induced depolarization and an increased sensitivity to alpha 1-adrenergic stimulation. No differences were noted regarding the Ca2+ sensitivity of force during K(+)-induced depolarization. The results suggest that microvascular growth (1) is immediately and positively influenced by the organ growth; (2) results in a functional resetting of the microvascular segments towards larger diameters without gross morphological or mechanical alterations; and (3) is accompanied by pharmacological alterations of the smooth muscle reactivity.

Relaxant effect of phalloidin on Triton-skinned microvascular and other smooth muscle preparations.

Guinea pig mesenteric microarteries (diameter 60-100 microns), the main branch of the mesenteric artery and taenia coli were skinned with 1% Triton X-100 for 4 h at 4 degrees C. Microarteries, mounted for circumferential force measurement, developed maximal active force in response to elevation of the free Ca2+ (pCa = 4.52, in EGTA buffer) in the presence of ATP (7.5 mM) and calmodulin (0.1-0.3 microM). In these preparations, addition of phalloidin (1-100 microM) slowly (greater than 1 h) relaxed submaximal contractions (pCa greater than 4.52) in a dose-dependent manner. Relaxation was irreversible as, after phalloidin wash-out, subsequent active force to pCa = 4.52 was also reduced. By contrast, phalloidin preincubation and wash-out under relaxed conditions (pCa greater than 8) only reduced subsequent force to pCa = 4.52 on prolonged stimulation. The extent of phalloidin-induced relaxation was not dependent on free Ca2+ between pCa 6.40 and 4.52. Phalloidin-induced relaxation did not occur during rigor contractions (i.e. absence of ATP and Ca2+). These mechanical effects of phalloidin were accompanied by a decreased leak of actin out of the skinned preparations and by the prevention of guanidine extraction of actin from these preparations. Phalloidin did not inhibit the myosin light chain kinase or phosphatase activity isolated from these preparations. In addition, the relaxant effects were also noted in taenia coli and the main branch of the mesenteric artery but not in skinned porcine ventricular heart muscle. These experiments suggest the possible participation of actin filament dynamics on the maintenance of active force in Triton-skinned smooth muscle.

Higher Ca2+ sensitivity of triton-skinned guinea pig mesenteric microarteries as compared with large arteries.

Small mesenteric resistance arteries and the main branch of the mesenteric artery (outer in situ diameter 115 +/- 3 microns [n = 76] and greater than 1,000 microns, respectively) were skinned with 1% Triton X-100. Both preparations were mounted as rings for circumferential force measurement in an EGTA solution (free Ca2+, less than 10 nM; calmodulin, 0.3 microM; pH 6.7). Force-pCa curves were obtained by increasing free Ca2+ (0.05 to 30 microM). The resulting dose-dependent contractions, after normalization to maximal force development (arteries, 21.4 +/- 2.4 [n = 3]; arterioles, 15.3 +/- 2.1 mN/mm2 [n = 5]) were fitted to sigmoidal force-pCa curves. Values of ED50 and of the cooperativity factor h were 6.08 and 2.39 in arterioles and 5.64 and 1.64 in arteries. The higher Ca2+ sensitivity of arteriolar preparations remained at pH 7.0 at higher calmodulin concentrations and after inhibition of smooth muscle phosphatase with okadaic acid. Total myosin light chain kinase activity in crude arteriolar extracts (using [gamma-32P] ATP and isolated gizzard light chains as substrates) was approximately 25% of arterial kinase. Both kinase preparations had identical Ca2+ sensitivities. Likewise, total arteriolar phosphatase activity (using 32P-labeled gizzard light chains) was approximately 25% of the arterial activity; both phosphatases had an identical sensitivity toward okadaic acid. The ratio of kinase/phosphatase activities was identical in both tissues. Extracts of both tissues contained two isozymes of the myosin heavy chain as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential skinning of smooth muscle: a new approach to excitation-contraction coupling.

In intact smooth muscle, the calcium sensitivity of force may increase as well as decrease. Using differently skinned smooth muscle preparations, two pathways involved in the change of calcium sensitivity of force have been identified: in microarteries skinned with beta-escin, GTP (possibly via a G protein) is involved in sensitization to Ca2+, while in porcine coronary arteries skinned with Triton X-100 cyclic AMP and possibly cyclic GMP are involved in desensitization. In both cases, there is a corresponding change in the phosphorylation of the regulatory light chain of myosin, suggesting that the balance of phosphorylating to dephosphorylating reactions is altered at constant [Ca2+].

Effects of beta-antagonists on contraction of bovine retinal microarteries in vitro.

Contractile responses of bovine retinal microarteries (BRA) (diameter: 198 +/- 5 microns, n = 49) to beta-antagonists, local anesthetics and Ca2(+)-antagonists were studied in vitro. Propranolol (10(-8)-10(-5) M) relaxed K(+)-activated BRA dose-dependently, whereas timolol (10(-8)-10(-5) M) relaxed K(+)-activated BRA only weakly at the highest doses. The relaxation by propranolol was not mediated through interaction with adrenergic nerve endings, since fluorescence histochemistry showed absence of such nerve endings in BRA. In addition, propranolol still relaxed BRA which were treated with 6-hydroxydopamine (6-OHDA), which causes chemical adrenergic denervation. Local anesthetic properties of propranolol had no part in the relaxation: lidocaine (10(-7)-10(-5) M) did not relax K(+)-activated BRA. Verapamil (10(-9)-10(-6) M) relaxed K(+)-activated BRA markedly and dose-dependently. Both verapamil and propranolol relaxed phasic K(+)-induced force more than tonic force. By contrast, they relaxed only the tonic part of serotonin-induced force, and they had no effect on stretch-induced active force. Therefore: 1) propranolol dilates BRA more than does timolol, possibly because of the Ca2(+)-antagonistic properties of the former; 2) beta- and Ca2(+)-antagonists probably spare myogenic autoregulation of blood flow and do not prevent, but could partially reverse, serotonin-induced arterial spasm.

Mechanics of K(+)-induced isotonic and isometric contractions in isolated canine coronary microarteries.

The effects of shortening in isotonic contractions on the mechanics of microvascular smooth muscle were investigated. Intramyocardial canine coronary microarteries (in situ diameter 60 +/- 3 microns) were mounted as rings, connected to a newly developed photoelectromagnetic force-length transducer, and activated with 125 mM K+. Shortening during isotonic contractions depressed the length-force relation (shortening deactivation) compared with the length-force relation obtained from isometric contractions; the effect was present at the earliest moments after activation, suggesting that a fundamental mechanism associated with the actual sliding of contractile filaments delayed onset of contractile activity in isotonic contractions compared with isometric contractions. Force-velocity relations were obtained by isotonic quick releases from isotonic and isometric contractions at various times. Isotonic shortening before the quick releases reduced the constants of the apparent hyperbolic force-velocity relations and maximal velocity of shortening (Vmax) compared with isometric contractions released at the same time. Increasing contraction duration reduced Vmax but more so in isotonic than in isometric contractions. Vmax also decreased with decreasing instantaneous length. A possible effect of force development on Vmax before the isotonic quick release was also described. Quick increments of load during isotonic contractions were sustained during active shortening in the phasic part, but during the tonic part loading resulted in a pronounced transient relaxation. Thus, in microvascular preparations, active isotonic shortening altered the length-force, force-velocity, and velocity-time relations and uncovered a time-dependent sensitivity to loading conditions. These experiments suggested that the mechanics of smooth muscle contraction may contribute significantly to the mechanisms of the physiological control of coronary microvascular diameter.

Effect of alpha-1 and beta agonists on contraction of bovine retinal resistance arteries in vitro.

Contractile responses of bovine retinal arteries (BRA) (diameter: 179 +/- 9 micron, n = 25) to high K+, circumferential stretch and adrenergic stimulation were studied in vitro. BRA could be activated by rapid circumferential stretch. Under resting conditions, phenylephrine consistently activated BRA at the highest dose of the drug used (10(-5) M). During K+- and stretch-induced activation, significant contractile responses to phenylephrine appeared at lower doses (respectively, 3.10(-8) and 10(-6) M). Isoproterenol did not relax K+- and stretch-induced contractions. Therefore, (1) BRA probably can autoregulate through a myogenic mechanism on the basis of stretch; (2) during alpha 1 adrenergic stimulation, myogenic autoregulatory responses probably increase; (3) contractile responses to alpha 1 adrenergic stimulation are masked under resting conditions; and (4) BRA may not possess functional beta adrenergic receptors.

Positive inotropic and vasodilating effects of amrinone and milrinone in isolated canine heart.

The question has been raised whether the in vivo positive inotropic effect of amrinone and milrinone is a primary effect or secondary to vasodilation. The effects of each drug on isolated trabeculae and resistance vessels obtained from the same dog hearts were determined separately. The positive inotropic and vasodilatory effects coincided over the same concentration range for amrinone. The active isometric force of trabeculae and resistance vessels was increased, and respectively decreased by 20% at comparable concentrations of amrinone--20% ED (effective dose) ranging between 9.5 and 18 microM. By contrast, the vasodilatory properties of milrinone (20% ED, 15-34 microM) appeared only at concentrations at which milrinone had already evoked a maximal positive inotropic response in trabecular muscle (20% ED, 0.36-0.38 microM). Based on the present experiments and their limitations, it would thus appear that, in the intact heart and at therapeutic doses, positive inotropic and vasodilatory effects may be equally present for amrinone, whereas, for milrinone, the positive inotropic effect would largely predominate the vasodilatory effect.

Moderate cooling depresses the accumulation and the release of newly synthesized catecholamines in isolated canine saphenous veins.

Moderate cooling (from 37 degrees to 24 degrees C) depressed the formation of 3H-dopamine and 3H-norepinephrine from 3H-tyrosine by isolated canine saphenous veins. Cooling reduced the evoked release of newly synthesized catecholamine to the same extent as that of stored norepinephrine. Hence the augmentation by cold of the contractile response to sympathetic nerve stimulation observed in earlier work is not accompanied by an augmented release of newly synthesized norepinephrine.

Tissue accumulation and release of newly synthesized 3H-dopamine and 3H-noradrenaline in canine saphenous veins incubated with 3H-(--)-tyrosine.

Experiments were designed to study the release of newly synthesized catecholamines in the isolated canine saphenous vein. Unlabelled (--)-tyrosine did not affect the contractions caused by electrical stimulation or exogenous noradrenaline and did not influence the basal efflux and the stimulation-induced overflow of 3H-noradrenaline in veins, previously incubated with the radiolabelled transmitter. The precursor increased the stimulation-induced overflow of the principal intraneuronal metabolite of 3H-noradrenaline, 3H-3,4-dihydroxyphenylglycol, and augmented the concentration of endogenous dopamine. Available column chromatographic procedures were modified to measure small amounts of 3H-catecholamines in the presence of large concentrations of tritiated (--)-tyrosine. Incubation of isolated veins with 3H-(--)-tyrosine caused concentration- and time-dependent tissue accumulation of newly synthesized catecholamines; the amounts of 3H-noradrenaline and 3H-dopamine were roughly comparable and were augmented by raising the external K+ concentration. In isolated veins, first incubated with 3H-(--)-tyrosine, and then superfused, a small basal efflux of 3H-noradrenaline was detected. Electrical stimulation caused a frequency-dependent overflow of 3H-catecholamines consisting mainly of 3H-noradrenaline; a stimulation-evoked efflux of 3H-dopamine (10% of total 3H-catecholamines) could be detected only when long stimulation periods were applied. After a 70 min period of superfusion, the 3H-dopamine content of the tissues decreased while that of 3H-noradrenaline remained unchanged, irrespective of whether the tissues had been stimulated electrically or not. The present results show that the isolated canine saphenous vein can synthesize and release 3H-noradrenaline and 3H-dopamine when incubated with 3H-(--)-tyrosine.