Traditionally, discussions of the roles of Ca+ + in vascular smooth muscle function have emphasized the direct relationship between changes in intracellular free Ca+ + levels and vascular contractility. However, as stated previously, the primary step regulating intracellular free Ca+ + levels is the accumulation, binding, and release of Ca+ + . Qualitative and quantitative variations observed in vessel responsiveness may be attributed to differing Ca+ + permeabilities and binding properties in each type of vascular smooth muscle in a given species. Characterization of Ca+ + kinetics in vascular muscles must, therefore, be based upon approaches designed to dissociate differing Ca++ mobilization and sequestration mechanisms and to compare the relative importance of each one in specific vascular systems.
In recent years, several technical approaches have been developed to facilitate dissociation of Ca+ + components in vascular smooth muscle. The principal techniques employed and the related specific cellular components that can be identified are listed in Table 1. In combination, these measurements provide a logical experimental sequence for determining: (a) the relative importance of various Ca+ + accumulation and mobilization mechanisms present in a particular type of vascular smooth muscle (Fig 1), and (b) the primary mechanisms by which vasoactive agents affect mobilization of Ca* + in each vessel. The initial experimental step is to measure the degree of dependence of contractility upon extracellular Ca++. In rabbit aorta, the actions of norepinephrine on a phasic component, resistant to depletion in Ca++-deficient solutions, can be dissociated from the high K+-induced response that is rapidly abolished in the absence of extracellular Ca++. This indicates the existence of two distinct Ca+ + components. However, in other vessels, more complex procedures are necessary because some of the cellular Ca+ + components are either more labile (and more readily depleted in Ca+ +-deficient solutions) or of smaller magnitude.
The most informative subsequent experiment is to measure Ca++ retention after washout of tissues in cold La+ + +-containing solution. This analytical technique removes extracellular Ca++ (80 to 90 percent of the total Ca++) and permits estimation of relatively small changes in cellular Ca+ + in the absence of large nonspecific Ca+ + components and significant transmembrane Ca+ + fluxes during the washout period. Plotting of Ca++ uptake values at different extracellular Ca+ + concentrations on Scatchard-coordinates identifies those extracellular Ca++ concentrations that, in each vessel, are appropriate for measurement of predominantly high or low affinity Ca++ components. Furthermore, since selected concentrations of Sr+ + can specifically displace high affinity Ca+ + without altering low affinity Ca+ + uptake, use of Sr+ + can demonstrate a parallel block of Ca++ release and the effects of associated tension responses. This can be particularly valuable in situations where bound Ca++ fractions are labile. Canadian Neighbor Pharmacy represents a new project. o know more you may here – http://treatment-cnp-online.nl/.
Finally, use of Ca++ efflux measurements and microsomal membrane preparations to ascertain whether bound Ca+ + is directly altered by vasoactive agents provides important mechanism-related information. Even though smooth muscle microsomal preparations contain both plasma membranes and sarcoplasmic reticulum, it is possible with these preparations to distinguish between agents that are direct Ca+ + antagonists (eg, aminoglycoside antibiotics) and those acting indirectly at other sites to alter uptake or mobilization of Ca++(eg, Ca++ entry blockers).
As mentioned previously, in rabbit aortic smooth muscle the Ca+ + fractions responsible for K+-induced Ca+ + uptake are very dissimilar to those of norepinephrine-induced Ca+ + release in regard to both dependence on extracellular Ca* * and Ca++ kinetics. Thus, these fractions can be readily dissociated. Delineating the different Ca* +components present may be more difficult in other vascular smooth muscle preparations if: (a) the higher affinity component is more labile than that in rabbit aorta, and the resulting Ca+ + kinetics (and Ca+ + depletion in Ca++-deficient solutions) are more rapid, and (b) the relative importance of various components for induction and maintenance of contractility differs from corresponding components in rabbit aorta. In a recent review article, the distinct voltage-sensitive and receptor-linked Ca+ + entry channels in aorta were contrasted with the only partial functional separation of these two channel types in most vascular muscles studied.
If the above considerations are applied to studies of different vessels, the presence of similar (but less readily separated) Ca++ mobilization components can be demonstrated. For example, in canine coronary arteries, the responses elicited with high K+ were found to be more sensitive to Ca++depletion than were those obtained with the agonist prostaglandin F*,. In the same study, the low affinity Ca++ uptake after washout in cold La+ ++–containing solution was increased by high K+ but not by prostaglandin F. Conversely, the corresponding high affinity Ca++ uptake was decreased by prostaglandin F, but not by high K+. Thus, in the canine coronary artery, the actions of high K+ and prostaglandin F are qualitatively similar to those of K+ and norepinephrine, respectively, in rabbit aortic smooth muscle.
Qualitatively different relationships in other vessels can also be obtained with similar experimental approaches. An example of this was described in a study comparing canine renal arteries and veins. Apparently, the La+ ++-resistant high affinity Ca++ component is present to a much larger extent in canine renal arteries than in canine renal veins. In rabbit aortic smooth muscle, this component is mobilized by norepinephrine to elicit tension responses. By analogy, the canine renal artery should be more sensitive to stimulation by norepinephrine than is the canine renal vein. However, sensitivity of contractile responses to norepinephrine was similar in both types of vessel. Examination of “Ca uptake and release by norepinephrine (10~e M) reveals different patterns of Ca+ + mobilization in canine renal arteries and veins. In the arteries, norepinephrine releases Ca++ from a high affinity store, whereas norepinephrine increases Ca+ + uptake in the veins. Thus, under similar experimental conditions, qualitatively different mechanisms for Ca++ mobilization are activated by norepinephrine.
Some studies, mainly with rabbit main pulmonary artery, indicate that pulmonary arterial smooth muscles also can mobilize Ca+ + from different sources. In rabbit main pulmonary artery, K+-induced contractile responses are more dependent on extracellular Ca++ than are responses to other agonists such as norepinephrine, prostaglandin F, and acetylcholine. Uptake of Ca++ is increased by physiologically relevant concentrations of norepinephrine without significant depolarization, but larger tension responses to higher concentrations of norepinephrine could be correlated with mobilization of cellular Ca++ stores. Tetraethylammonium chloride (TEA) elicited tension responses in rabbit main pulmonary artery by increasing membrane permeability to Ca+ + in a manner similar to that observed with high K+. Both the K+-induced responses in guinea pig pulmonary artery and hypoxic pulmonary vasoconstriction in rat lungs were inhibited by voltage-sensitive Ca++ entry blockers. Thus, pulmonary arterial smooth muscles appear to mobilize Ca++ by mechanisms similar to those delineated in other vessels.
In vascular smooth muscle systems, Ca++ can be mobilized from extracellular, membrane, and intracellular sources. A general model has been proposed in which Ca+ + can be present at both superficially located and cellular high and low affinity sites and can enter the cell by at least three routes (resting entry, voltage-sensitive entry, receptor-linked entry). Similar Ca+ +-related components appear to be present in different types of vascular smooth muscles, but their relative importance in induction and maintenance of contractile tone and tension can differ. Measurement of tension responses under differing conditions does not provide sufficient information for unequivocal identification of critical Ca+ + sources. However specific techniques have been used to delineate Ca++ mobilization mechanisms in a number of isolated vascular preparations. These approaches can be applied in a systematic manner to pulmonary vessels to identify those Ca++ stores and uptake mechanisms of particular importance for responses to agonists and antagonists.
Figure 1. Model for different Ca+ + uptake pathways and associated Ca+ + binding sites in vascular smooth muscle.
Table 1—Approaches Useful in Dissociation and Measurement cf Ce++Components in Vascular Muscle
|Technique||Component Measured||Information Obtained|
|Tension||Dependence on||Show relative|
|extracellular Ca+ +||various Ca+ +|
|Wash in cold||Cellular Ca* +||Measure small|
|La+ + +||changes in|
|solution||intracellular Ca+ +|
|Scatchard-||Cell Ca++ fractions||Determine presence|
|coordinate||of high and low|
|plot||affinity Ca+ + components|
|Sr++–||High affinity Ca+ +||Assess degree of|
|solutions||agonist action on high affinity Ca* *|
|Ca++ efflux||*®Са washout||Estimate effects on Ca++ exchange and rate of loss|
|Microsomal||Binding and uptake of||Determine whether|
|Ca+ +||membrane Ca+ +||agonists directly|
|accumulation’||alter Ca+ + binding|