Isolation of Plasma Membranes from Rat Mesenteric Veins: A Comparison of Their Physical and Biochemical Properties with Arterial Membranes

Abstract

Plasma membranes were isolated from smooth muscles of rat mesenteric veins. The plasma membrane fraction is relatively pure according to its morphological and enzymatic characteristics. The membrane distribution, enzymatic activities, as well as calcium accumulation by the plasma membrane fraction from venous smooth muscle were compared to those from arterial smooth muscle. The isolated venous smooth muscle plasma membranes formed primarily closed vesicles which were capable of accumulating Ca²⁺ in the presence of ATP suggesting active transport of Ca²⁺ across the membrane vesicles. Evidence obtained from several approaches by studying the effect of A23187, phosphate ions and hypotonic shock on the Ca²⁺ accumulation in the presence of ATP revealed that there is an active transport of Ca²⁺ across isolated vascular smooth muscle membrane vesicles in addition to binding of Ca²⁺. However, venous smooth muscle plasma membrane fraction appears to be different from arterial smooth muscle plasma membrane fraction in its lower activity of alkaline phosphatase, greater Ca²⁺ binding and lower Ca²⁺ transport. These and previous studies show that the plasma membrane of vascular muscles may play an important role in the steady state regulation of cellular calcium concentration during excitation-contraction coupling, especially in small arteries and veins.

ATPase activity and calcium uptake of microsomes isolated from stomach smooth muscle after exposure to phospholipase C.

“…Although PLC treatment inhibited both Ca2+ , Mg2+-ATPase and ATP supported Ca2+ uptake of isolated microsomal fractions, a significant amount of Ca2+ uptake was resistant to PLC treatment even after 60min preincubation . It is possible, however, that the residual ATP supported Ca2+ uptake seen after 60min PLC treatment might have been due to ATP dependent binding which was substantially higher than Ca2+ binding in the absence of ATP (Kwan et al ., 1981), since binding of Ca2+ by microsomal fractions was slightly but not significantly affected by PLC treatment ( Table 2).…”

“…Although a heterogenous microsomal fraction was used in this study , the biochemical (Hurwitz et al, 1973;Kwan & Ramlal, 1982) and (c) most smooth muscles have sparse content of endoplasmic reticulum (Devine et al, 1972) and the distribution of ATP supported Ca2+ uptake parallels that of plasma membrane markers in isolated subcellular membrane fractions from various types of smooth muscles (Janis et al, 1977;Grover et al, 1980;Kwan et al, 1981) including stomach smooth muscles (Sakai et al, 1981;Kawn et al, 1982). Our present study shows that PLC is a useful tool for study of the functional role of membrane phospholipids in membrane associated enzymes and ion transport systems and the results are consistent with our previous report that PLC treatment causes smooth muscle cell membrane damage leading to inhibition of smooth muscle activity.…”

“…Such Ca2+ transport systems have been reported to be associated with isolated crude microsomal fractions (Carsten & Miller , 1977;Wuytack et al, 1978) or highly purified plasma membrane fractions (Grover et al, 1980;Kwan et al, 1981;Sakai et al, 1981). It is known that phospholipase C (PLC) inhibits action potential, simultaneously depolarizing resting membrane potential; and affects the ionic permeability of neuron and muscle cell membrane (Tobias, 1958;Albuquerque & Thesleff, 1967;Sakai, 1980) .…”

Mg^2+- or Ca^2+-Activated ATPase Activities of Plasma Membranes Isolated from Vascular Smooth Muscle

“…The trimming procedure for the isolation of smooth muscle of mesenteric arteries and veins from the surrounding tissues, homogenization conditions and the subsequent isolation of plasma membrane fraction by differential centrifugation as well as su crose density gradient centrifugation has previously been described in detail [11]. When various subcellu lar fractions were employed, their definitions were as follows: postnuclear supematent (PNS) was obtained as the supematent after centrifugation of tissue ho mogenate at 900 g for 10 min; mitochondrial fraction (MIT) was obtained as a pellet by centrifugation of PNS at 9,000 g for 10 min.…”

“…Such microsomal ATP hydrolysis is also effectively activated by Ca2+ [3,4], Ca2+-stimulated, Mg2+-dependent ATPase activity in the microsomal fraction from several vas cular smooth muscle preparations has also somal membrane fractions isolated from large arteries which are structually and func tionally different from small arteries and veins. Although highly enriched plasma membrane fraction and other relatively het erogeneous subcellular membrane fractions have recently been isolated from rat mesen teric arteries and veins for the study of the characteristics of ATP-dependent Ca2+ trans port in vascular smooth muscle under nor mal [10][11][12][13] as well as pathophysiological condition [12], there is yet little information concerning the general properties of such 'basic' ATPase in small arteries and veins. In this study, the following questions are inves tigated: What is the subcellular origin of such Mg2+-or Ca2+-activated ATPase activity?…”