Calcium-accumulating properties of subcellular fractions of bovine vascular smooth muscle.

Abstract

This study examined the calcium transport properties of two subcellular fractions of bovine aorta obtained by differential centrifugation. The vesicular fraction had a high-affinity (K m = 1.05 x 10~6M) calcium transport mechanism which could be potentiated by using the calcium-precipitating anion, oxalate. The mitochondriaenriched fraction's calcium transport system had a lower affinity for calcium than did that of the vesicular fraction. The calcium capacity of the vesicular fraction was determined by comparing the steady-state calcium uptake in the presence of oxalate in the whole homogenate with the same uptake by the vesicular fraction alone. A calcium capacity of about 100 /imoles Ca 2+ /kg aorta was obtained. It is concluded that the vesicular fraction has all of the properties of a major site of subcellular calcium regulation in vascular smooth muscle.• It is generally accepted that intracellular free calcium is the connection between stimulation and contraction in vascular smooth muscle (1, 2). However, the identification of the sources and sinks of the activator calcium remains an unsettled question. Numerous indirect experiments have shown that at least part of the activator calcium is intracellular in origin (1-4). Recent electron microscopic studies indicate that there are at least two different subcellular organelles potentially capable of serving as intracellular sinks for calcium: one is the mitochondria and the other is the sarcoplasmic reticulum (5-7). Both of these structures are capable of sequestering the divalent ion strontium in contracting muscle, and the mitochondria can also sequester barium. To determine if these or any other subcellular structures are capable of playing major roles in the regulation of the intracellular calcium concentration of vascular smooth muscle, numerous questions must be answered. Are these structures capable of accumulating calcium at the very low cytoplasmic level (10~5M) sufficient for maximal activation of the contractile process (8)? If they are capable of sequestering calcium at this low level, what is the relative competitive position of each structure? Can these structures, alone or in

Ca⁺⁺-Binding Properties of Canine Aortic Microsomes: Lack of Effect of c-AMP

“…The addition of the inorganic anions, oxalate and phosphate, which are known to stimulate calcium sequestration in both cardiac and skeletal muscle by precipitating calcium within the vesicles [21], had no effect on the aortic microsomes. The lack of oxalate stimulation differs from the results obtained with aortic microsomes by others [11,12,14,15] who showed significant stimulation by these agents. Basically the same isolation technique is used here as described by F itzpatrick el al.…”

“…[15]. D 'A uriac et al [8], F ord and H ess [12] and H ess and F ord [14] used imidazole buffer. as indicated in the absence of any A number of other compounds reported to affect calcium binding or release in cardiac relaxing system did not affect the aortic microsomes.…”

“…In the past few years, a number of investigators have successfully isolated a sarcoplasmic reticular fraction from various sources of smooth muscle [1,4,6,8,11,12,14,15] and have demonstrated ATP-dependent Ca++ binding to these subcellular fractions. These developments have been extremely important since they offer biochemical evidence that sug gests some aspects of similarity of relaxation mechanisms in cardiac, skeletal and smooth muscle types.…”

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

“…In initial microsomal studies [Katz and Repke, 1967;Harigaya and Schwartz, 1969;Ford and Hess, 1975], Ca++ incorporation in the presence of both ATP and oxalate was designated as Ca++ 'uptake'. Ca++ incorpora tion in the presence of ATP but not of oxalate was designated Ca++ 'binding'.…”

Possible role of phosphorylation-dephosphorylation in the regulation of calcium metabolism in cardiovascular tissues of SHR.

“…18 -19 '"-" From studies on microsomal fraction of smooth muscle, Ford and Hess 28 concluded that the microsomes sequestered sufficient Ca 2+ and that this fraction has the capability to be both sink and source for the activator Ca 2+ in excitation-contraction coupling. Observations made in this laboratory" and by others 8 "' demonstrate that incubation of smooth muscle microsomal fraction with cAMP-dependent protein kinase enhances energy-dependent Ca l+ sequestration, which suggests a modulatory role for cAMP-dependent protein kinase in Ca 2+ transport in vascular smooth muscle.…”