Protein kinase C translocation in intact vascular smooth muscle strips

Abstract

Using intact muscle strips from the bovine carotid artery, the time course of translc~ation of protein kinase C (PKC) from the cytosol to the membrane fraction was measured in response to various agonists at induce contractile responses. PKC activity was assessed by Ca2+/phospholipid-dependent phosphorylation of histoiie. Exposure of the muscle strips to phorbol ester (12-deoxyphorbol 1 3-isobutyrate) induced a rapid and sustained translocation of PKC from the cytosol to the membrane fraction, and a slowly developing but sustained contractile response. Histamine induced a comparable initial translocation of PKC to the membrane which then decreased somewhat to a stable plateau significantly above basal values. Histamine also led to a rapid and sustained increase in tension. Angiotensin I, which caused a rapid but transient contraction, induced a rapid initial translocation of PKC to the membrane. The men. brane-associated PKC then declined to a stable plateau significantly lower than that seen after a histamine-induced respon.e, and only slightly above the basal value. Endothelin, which induced a sustained contraction, caused a sustained translocation of PKC from the cytosol to the membrane. In contrast, although exposure to 35 mM-KCI induced a rapid and sustained contraction, it caused only a transient translocation of PKC; the membrane-associated PKC returned to its basal value within 20 min. These results demonstrate that PKC in intact smooth muscle can be rapidly translocated to the membrane and remains membranebound during sustained phorbol ester-or agonist-induced contractions, but that such .t sustained translocation of PKC does not occur during prolonged stimulation with KCI. INTRODUCTIONThe Ca2+/phospholipid-dependent protein kinase, protein kinase C (PKC), is widely distributed in tissues and organs [1]. Over the last few years the enzyme has been implicated in the regulation of an increasing number of cellular processes [1][2][3][4]. In vascular smooth muscle, PKC is present in relatively high concentrations [4], suggesting that it also plays an important role in the control of smooth muscle function. A unique feature of PKC regulation is the spatial translocation of the enzyme. Under basal conditions PKC is thought to be located mainly in the cytosol, but after hormonal stimulation PKC is rapidly translocated to the membrane where it associates with membrane phospholipids [4]. The activation of the enzyme is often closely linked to changes in phosphoinositide (PI) metabolism [3]. When an appropriate agonist activates a specific receptor, an immediate receptor-linked event is the activation of a specific phospholipase C that catalyses the rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to two intracellular messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [2]. It has been shown that IP3 triggers the release of Ca2+ from intracellular stores in smooth-muscle [5]. Likewise, it has been demonstrated that appropriate agonists induce sustained increases in the DAG cont...

Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle

“…A low concentration of DPB (100 nM) was also used to modulate high K+-induced contractions. Measurement ofprotein kinase C activity Cytosolic and membrane fractions were prepared from rat aorta as described previously (Haller et al, 1990). After a 20 min incubation with test agents, muscle strips were quickly frozen in liquid nitrogen and crushed.…”

Endothelium‐derived relaxing factor inhibits the endothelin‐1‐induced increase in protein kinase C activity in rat aorta

“…For example, prolactin stimulation of rat aortic smooth muscle has been shown to lead to simultaneous increases in both particulate and cytosolic PKC activity (Sauro et al, 1989). In contrast, agonist-induced decreases in cytosolic PKC activity with corresponding increases in particulate PKC activity have been shown by a number of workers (WheelerJones et al, 1989;Chambers et al, 1990;Haller et al, 1990). An unusual situation occurs in platelets in that either dual activation of both cytosolic and particulate enzymes can occur or, alternatively, the cytosolic activity decreases while the particulate activity increases depending on which aggregating agent is used (Salari et al, 1990 The lack of any real association between increases in particulate PKC activity and decreases in cytosolic PKC activity in the presence of endothelin-1 leads us to suggest that in this model, the two pools of the PKC isoenzymes may be acting independently of one another.…”

“…One such study (Haller et al, 1990) (Nishizuka, 1984) are also of importance. The DAG response of vascular smooth muscle to endothelin-1 has been studied by a number of groups.…”

“…Several constrictor agonists including endothelin have now been shown to cause activation and translocation of PKC from the cytosolic to the particulate fraction of vascular smooth muscle cells (Haller et al, 1990) and it has been shown that these events are responsible for the maintenance of tone during the tonic phase of contraction.…”

“…In vascular smooth muscle, protein kinase C (PKC) is present in relatively high concentrations (Nishizuka, 1984) and is presumed to play an important role in the regulation of tone, particularly during the tonic phase of contraction (Rasmussen et al, 1987;Haller et al, 1990). Several constrictor agonists including endothelin have now been shown to cause activation and translocation of PKC from the cytosolic to the particulate fraction of vascular smooth muscle cells (Haller et al, 1990) and it has been shown that these events are responsible for the maintenance of tone during the tonic phase of contraction.…”