George B. Weiss

Effects of newly arachidonic acid metabolites on microsomal Ca++ binding, uptake and release

The 5,6-; 8,9-; 11,12- and 14,15-epoxyeicosatrienoic acids and their respective hydration products, the vic-diols, recently reported as metabolites of arachidonic acid in rat liver microsomes, were examined for effect on release of 45Ca from canine aortic smooth muscle microsomes. At 10(-6) M, the diols had no effect, but the 5,6-; 11,12- and 14,15-epoxyacids increased the loss of 45Ca. Further studies with the 14,15-epoxyacid demonstrated a dose-dependent decrease of Ca++ uptake (ATP present) in canine aortic microsomes in 0.03 mM Ca++, whereas Ca++ binding (ATP absent) was not affected. Ca++ uptake, binding and release in rat liver microsomes was similarly affected by the 14,15-epoxyacid, the major epoxyeicosatrienoic acid derivative produced by rat liver microsomal incubations. It is suggested that alterations in Ca++ metabolism might be a possible mechanism of action for these derivatives of arachidonic acid.

Inhibitors of mitochondrial Ca++ uptake dissociate potassium-induced tension responses from increased 45Ca retention in rabbit aortic smooth muscle.

Effects of antimycin A, oligomycin and KCN on tension responses to high K+ or NE and on related Ca++ movements were investigated in rabbit aortic smooth muscle. Selected concentrations of antimycin A (10(-6) g/ml), oligomycin (10(-6) g/ml) and KCN (1 mM) had little effect on the K+-induced contractile response even though all three agents abolished the increase in La+++-resistant low affinity Ca++ uptake elicited with high K+. Though the mitochondrial inhibitors had varied effects on the NE-induced contractile response, they had no effect on the NE-induced decrease in La+++-resistant high affinity Ca++. Oxygen consumption of the muscle tissue was decreased by antimycin A or oligomycin. These results suggest that the increase in low affinity Ca++ retention elicited with high K+ represents Ca++ uptake by mitochondria. Thus, the correlation between K+-induced uptake of Ca++ and an increased retention of La+++-resistant low affinity Ca++ after exposure to K+ are sequential events that can be associated by inhibitors of mitochondrial Ca++ uptake.

Quantitative Measurement of Binding Sites and Washout Components for Calcium Ion in Vascular Smooth Muscle

In a recent review article (Weiss, 1977a), a general hypothesis was developed concerning the central importance of Ca2+ to any comprehensive understanding of responsiveness of muscle systems to pharmacological agents. Briefly, this view was that both the qualitative and the quantitative nature of the muscle response to a given agent is directly determined by the amounts of intracellular Ca2+ available and this, in turn, is a direct consequence of differing Ca2+-binding properties of the specific cytoarchitecture present in each particular type of muscle. This is not to say that other phases of the sequence beginning with the drug-receptor interaction are not also essential. However, determination of the nature of the final response obtained is not a function of receptor activation but, rather, a result of alterations in membrane binding and permeability characteristics and, ultimately, a direct function of the intracellular Ca2+ level. Though this relationship exists in all types of muscle, elaboration of correlations between muscle tension and Ca2+ distribution and movements has not progressed as rapidly in smooth muscle as in striated muscle. The sequence of events leading from membrane excitation to muscle contraction in striated muscle has been convincingly documented (see Sandow, 1965; Bianchi, 1968) and related to clearly defined morphological structures. However, in smooth muscle anatomical structures resembling those observed in striated muscle are not as readily identified and, partly as a consequence of this, specific cellular sources and sites for Ca2+ cannot be delineated in a parallel fashion.

Effect of transmembrane pH gradient changes on potassium-induced relaxation in vascular smooth muscle.

In bicarbonate-buffered solution the Ba++-induced contraction of rat aorta was relaxed by an elevated extracellular K+ concentration. When the pH of the bicarbonate-buffered solution was lowered or the buffer system of the solution changed from bicarbonate to Tris or HEPES, the K+-induced relaxation was strongly inhibited. Raising the pH of the Tris-buffered solution restored the effect of K+. Addition of NH4+, which is known to cause a transient increase in intracellular pH, transiently inhibited and subsequently augmented the K+-induced vasodilation in all solutions. It is suggested that the K+-induced vasodilation results from membrane hyperpolarization following enhanced Na+, K+-ATPase activity and that this K+-induced vasodilation is affected by changes in the transmembrane pH gradient.

Qualitative differences in 45Ca efflux from membrane sites in vascular smooth muscle when washout conditions are varied

Abstract 1. 1. Rabbit aortic media-intimal strips were incubated with 45 Ca and washed out in solutions containing 1.5 mM Ca 2+ , no added Ca 2+ plus 0.05 mM EDTA, 1.5 mM Ca 2+ at 0.5°C, and isosmotic (80.8 mM) La 3+ at 0.5°C. 2. 2. Scatchard coordinate plotting of Ca 2+ remaining in the muscles after varied washout intervals in differing solutions indicates that dissimilar portions of La 3+ -accessible or La 3+ -inaccessible high or low affinity Ca 2+ components are altered. 3. 3. Knowledge of how washout variations affect different components can be critical for selection of appropriate experimental conditions for analysis of actions of drugs and ions on 45 Ca fluxes.

Effects of prostaglandin E1 on contractility and 45Ca release in rabbit aortic smooth muscle

Concentrations of prostaglandin E1 (PGE1; 10(-7) M) that do not elicit tension responses in aortic strips potentiate contractions induced by submaximal concentrations (10(-8)-10(-7) M) of norepinephrine (NE) or angiotensin III (Ang III) but not those of high K+ depolarization or maximal NE or Ang III concentrations. Higher concentrations of PGE1 (10(-6) M and above) initiate contractions which are additive with submaximal responses to NE and Ang III but not to K+. These same concentrations of PGE1 also decrease 45Ca retention at high affinity La+++-resistant sites in a manner similar to but not additive with NE and Ang III. Uptake of 45Ca at low affinity La+++-resistant sites (which is increased by high K+-depolarization) is not altered by 10(-6) M PGE1. The effects of PGE1 are not altered by decreased extracellular Ca++ (0.1 mM), decreased temperature, phentolamine or meclofenamate. Thus, PGE1 does not appear to increase uptake of extracellular Ca++ in this smooth muscle tissue. Instead, PGE1 increases mobilization of Ca++ from the same high affinity La+++-resistant sites affected by Ang III and NE and, in this manner, may increase responses to these two stimulatory agents.

Delineation of high and low affinity 45Ca incorporation and of 45Ca efflux in canine aortic smooth muscle microsomes

Abstract 1. 1. Scatchard plots of Ca 2+ incorporation in canine aortic media-intima microsomes indicate two types of binding sites. Low affinity binding sites equilibrate more rapidly. 2. 2. Ca 2+ incorporation is higher in a NaCl medium than in a KCl medium at a Ca 2+ concentration in which high affinity binding predominates. The high affinity 45 Ca binding is largely ATP-dependent. 3. 3. Efflux of non-superficial 45 Ca from the microsomes is linear during a 32-min washout into a solution containing no added Ca 2+ and is increased by addition of 0.1 mM EGTA, 1 mM Ca 2+ or 1 mM Sr 2+ .

Effects of manganese on 45Ca mobilization and contractile responses in rabbit aortic smooth muscle

In rabbit aortic smooth muscle, 1.5 mM Mn2+ inhibits contractile responses to norepinephrine more than those to high K+. Other associated effects include decreases in the La3+-resistant high affinity Ca2+ fraction, the high affinity Ca2+ uptake component in the Scatchard-coordinate plot, and high affinity Ca2+ uptake and binding in canine aortic microsomes. Higher (15 mM) concentrations of Mn2+ are less selective. Thus, those Ca2+ mobilization components and contractile responses associated with high affinity bound Ca2+ are more sensitive to inhibition by Mn2+.

Specific Alterations in Binding and Uptake of High and Low Affinity Ca++ in Canine Aortic Microsomes

The effects of Sr++, aminoglycoside antibiotics and other agents on Ca++ incorporation by canine aortic microsomes have been examined in 0.03 mM Ca++ (high affinity Ca++ components predominate) and in 3.0 mM Ca++ (low affinity Ca++ components predominate) in the presence of 45Ca uptake) and absence (45Ca binding) of ATP. Sr++ inhibited high affinity Ca++ uptake more than binding of Ca++ and reduced low affinity Ca++ uptake and binding only at higher concentrations (3.0-5.0 mM). Similar results were obtained with Sr++ in canine aortic media intimal strips. Neomycin (0.4-1.4 mM) or gentamicin (0.6-2.0 mM) had no significant effect on high affinity Ca++ uptake, but reduced high affinity Ca++ binding and inhibited low affinity Ca++ uptake and binding. Nitroprusside inhibited only high affinity Ca++ uptake and D-600 was without effect. Thus, measurement of Ca++ uptake and binding in smooth muscle microsomal membrane preparations demonstrates that agents affecting smooth muscle contractility can alter binding or uptake of Ca++ in differing ways or, in some cases (e.g. D-600), exert their action by blocking other Ca++-related effects.