Formation of nascent secretory vesicles from the trans-Golgi network of endocrine cells is inhibited by tyrosine kinase and phosphatase inhibitors.

Recent evidence suggests that secretory vesicle formation from the TGN is regulated by cytosolic signaling pathways involving small GTP-binding proteins, heterotrimeric G proteins, inositol phospholipid metabolism, and protein serine/threonine phosphorylation. At the cell surface, protein phosphorylation and dephosphorylation on tyrosine residues can rapidly modulate cytosolic signaling pathways in response to extracellular stimuli and have been implicated in the internalization and sorting of signaling receptors. to determine if phosphotyrosine metabolism might also regulate secretory vesicle budding from the TGN, we treated permeabilized rat pituitary GH3 cells with inhibitors of either tyrosine phosphatases or tyrosine kinases. We demonstrate that the tyrosine phosphatase inhibitors pervanadate and zinc potently inhibited budding of nascent secretory vesicles. Tyrphostin A25 (TA25) and other tyrosine kinase inhibitors also prevented secretory vesicle release, suggesting that vesicle formation requires both phosphatase and kinase activities. A stimulatory peptide derived from the NH2 terminus of the small GTP-binding protein ADP ribosylation factor 1 (ARF1) antagonized the inhibitory effect of TA25, indicating that both agents influence the same pathway leading to secretory vesicle formation. Antiphosphotyrosine immunoblotting revealed that protein tyrosine phosphorylation was enhanced after treatment with tyrosine phosphatase or kinase inhibitors. Subcellular fractionation identified several tyrosine phosphorylated polypeptides of approximately 175, approximately 130, and 90-110 kD that were enriched in TGN-containing Golgi fractions and tightly membrane associated. The phosphorylation of these polypeptides correlated with inhibition of vesicle budding. Our results suggest that in endocrine cells, protein tyrosine phosphrylation and dephosphorylation are required for secretory vesicle release from the TGN.

Role of the membrane skeleton in preventing the shedding of procoagulant-rich microvesicles from the platelet plasma membrane.

The platelet plasma membrane is lined by a membrane skeleton that appears to contain short actin filaments cross-linked by actin-binding protein. Actin-binding protein is in turn associated with specific plasma membrane glycoproteins. The aim of this study was to determine whether the membrane skeleton regulates properties of the plasma membrane. Platelets were incubated with agents that disrupted the association of the membrane skeleton with membrane glycoproteins. The consequences of this change on plasma membrane properties were examined. The agents that were used were ionophore A23187 and dibucaine. Both agents activated calpain (the Ca2(+)-dependent protease), resulting in the hydrolysis of actin-binding protein and decreased association of actin with membrane glycoproteins. Disruption of actin-membrane interactions was accompanied by the shedding of procoagulant-rich microvesicles from the plasma membrane. The shedding of microvesicles correlated with the hydrolysis of actin-binding protein and the disruption of actin-membrane interactions. When the calpain-induced disruption of actin-membrane interactions was inhibited, the shedding of microvesicles was inhibited. These data are consistent with the hypothesis that association of the membrane skeleton with the plasma membrane maintains the integrity of the plasma membrane, preventing the shedding of procoagulant-rich microvesicles from the membrane of unstimulated platelets. They raise the possibility that the procoagulant-rich microvesicles that are released under a variety of physiological and pathological conditions may result from the dissociation of the platelet membrane skeleton from its membrane attachment sites.

Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

Spindle cell lipoma of the foot and the application of CD34 immunohistochemistry to atypical lipomatous tumors in unusual locations.

Spindle cell lipoma demonstrates a distinctive histologic appearance and characteristic clinical presentation. We recently observed two cases of solitary subcutaneous neoplasm of the foot with histologic features of spindle cell lipoma that in one case includes a minor component of the overlapping tumor, pleomorphic lipoma. Because the foot is an unusual location for these neoplasms, immunoperoxidase and cytogenetic studies were performed. In both cases, staining was strongly positive for CD34 and negative for smooth muscle actin. Cytogenetic studies from the tumor with a pleomorphic component revealed features consistent with a lipomatous neoplasm, but are otherwise diagnostically nonspecific. An analysis of the literature reveals that although CD34 immunoreactivity is characteristic of spindle cell lipoma and helps exclude nonlipomatous neoplasms, it does not clearly eliminate other well-differentiated lipomatous tumors. Accordingly, without the aid of classic tumor location, the diagnosis of the spindle cell/pleomorphic lipoma group relies primarily on histologic features, with supportive but not definitive information provided by immunoperoxidase and cytogenetic studies. Obscuring this issue, however, are the imprecise histologic distinction between these tumors and those of the atypical lipoma/atypical lipomatous tumor/ well-differentiated liposarcoma group and the nomenclature controversy that surrounds the latter group of neoplasms. Despite these obstacles, both groups of well-differentiated lipomatous tumors are clinically benign when subcutaneously located.

The Veterans Administration Northwest Regional Health Services Research and Development Field Program: organization, activities, and early outcomes.

In 1982, the Veterans Administration established Health Services Research field programs in each of the six VA regions. Herein, we describe the historical origins, organization, responsibilities, activities, and early accomplishments of one of these programs--the Northwest Regional HSR&D field program. Special reference is made to this program's commitment to health services research relevant to geriatrics and gerontology, including the development of a system-wide agenda for research, information syntheses in geriatrics-relevant health services research topics, and the conduct of funded projects pertinent to care of the elderly. The importance of a medical center location for the field programs is discussed, and early indications of institutional impact are described.

Have we oversold case management as a "quick fix" for our long-term-care system?

Case management has become a core component in almost every community-based, long-term-care program. Yet it is, at best, a piecemeal response to an enormous health and social services problem. Fragmented funding, ill-considered public health policy, and short-range planning assure that no single system, even one as ubiquitous as case management, will eliminate the serious problems plaguing our health care system.

Case management in long-term care: options and opportunities.

Case management--emphasizing coordination of services to individual clients--has become a familiar component in the service delivery for community-based long-term care. The resource dependence theory of interorganizational relationships suggests guidelines for an expanded role that highlights case managers' allocation of resources and potential for intervention in local delivery systems.

Prosomatostatin processing in permeabilized cells. Calcium is required for prohormone cleavage but not formation of nascent secretory vesicles.

Our laboratory has been using a permeabilized cell system derived from rat anterior pituitary GH3 cells expressing prosomatostatin (pro-SRIF) to study prohormone processing and nascent secretory vesicle formation in vitro. Because calcium is necessary for prohormone processing enzyme activity, secretory granule fusion with the plasma membrane, and possibly sorting to the regulated pathway, we treated permeabilized cells with the calcium ionophore A23187 to determine the role of calcium in pro-SRIF cleavage and nascent vesicle formation from the trans-Golgi network (TGN). Here we demonstrate that pro-SRIF cleavage was markedly inhibited when lumenal free calcium was chelated with EGTA in the presence of A23187. Surprisingly, submillimolar free calcium (approximately 15 microM) was sufficient to maintain prohormone cleavage efficiency, a value far lower than that estimated for total calcium levels in the TGN and secretory granules. Experiments using both A23187 and the protonophore CCCP revealed that free calcium is absolutely required for efficient pro-SRIF cleavage, even at the optimal pH of 6.1. Secretory vesicle formation by contrast was not inhibited by calcium chelation but rather by millimolar extralumenal free calcium. Together, these observations demonstrate that pro-SRIF processing and budding of nascent secretory vesicles from the TGN can be uncoupled and therefore have distinct biochemical requirements. Interestingly, our data using intact GH3 cells demonstrate that basal secretion of SRIF-related material is largely calcium-dependent and therefore cannot be equated with constitutive pathway secretion. These results underscore the importance of determining calcium requirements before assigning a secretion event to either the constitutive or regulated secretory pathway.

Case management in the human services. Reflections of public policy.

One way to examine how case management has become a favored tool of public policy is to explore how it has evolved in various human service settings. This article compares, describes, and analyzes case management in six program categories: (a) for persons suffering from chronic mental illness; (b) in child welfare; (c) in community-based long-term care; (d) for children and adolescents suffering from severe emotional disturbance; (e) for individuals experiencing substance abuse problems; and (f) in the public welfare system. Case management in these settings is compared along three dimensions: legislative ad programatic origins; models; and outcome measures. The analysis reveals the extent to which case management has become a cornerstone of public policy--characterized by cost containment accomplished through substantial and continuing reduction in public support for human service programs.

Care planning in home care: an exploratory study.

Care Planning is one of the most important responsibilities of the case manager in home care. Research on how care plan decisions are made is scarce in the case management literature. The purpose of this exploratory study was to determine the factors that influence the decision-making process of case managers within the Edmonton Home Care Program (EHCP) when developing care plans. A volunteer sample of six EHCP case managers from three different disciplines was selected: two registered nurses (BScN), two occupational therapists (BScoT), and two social workers (BSW). With the use of fictional case studies, these professionals were asked to construct care plans. Individual semi-structured interviews and a group session permitted in-depth exploration of their decision-making process. Findings suggest that client and/or caregiver characteristics appear to be the most influential in determining care plans. It was also found that case managers are not comfortable with fiscal accountability.

Validating professional judgment in a home care agency.

In the absence of validate assessment techniques, health care practitioners often make professional judgments regarding their clients' needs for services. This article explores the validity and reliability of those judgments in a home care agency and discusses the implications for decision-making in long-term care.

The role of calpain in stimulus-response coupling: evidence that calpain mediates agonist-induced expression of procoagulant activity in platelets.

Although calpain (the Ca2(+)-dependent protease) is widely distributed, its function is poorly understood. One cell in which it becomes activated as a consequence of activation of the cell is the blood platelet. The aim of the present study was to determine whether activation of calpain was responsible for any of the responses of platelets to stimulation. Platelets were incubated with calpeptin, a membrane-penetrating inhibitor of calpain, before being exposed to an agonist. Concentrations of calpeptin that totally inhibited the agonist-induced hydrolysis of actin-binding protein (ABP) by calpain had no effect on many other responses associated with platelet activation: phosphorylation of myosin light chain, phosphorylation of P47, platelet shape change, aggregation of platelets, secretion of granule contents, or retraction of fibrin clots. However, these concentrations of inhibitor decreased the agonist-induced generation of procoagulant activity (assayed as the ability of platelets to catalyze the conversion of prothrombin to thrombin in the presence of factor Va and factor Xa). When thrombin was the agonist, the amount of ABP that was hydrolyzed was small; only a small component of the total agonist-induced procoagulant activity was inhibited by calpeptin. When collagen was the agonist, more ABP was hydrolyzed and the amount of procoagulant activity generated was greater; calpeptin decreased the collagen-induced procoagulant activity to levels comparable with those induced by thrombin in the presence of the inhibitor. We suggest that there are at least two mechanisms by which procoagulant activity is generated on activated platelets and that the agonist-induced activation of calpain mediates one of these mechanisms. These results show that activation of calpain is a component of the stimulus-response pathway in platelets.

Evidence that agonist-induced activation of calpain causes the shedding of procoagulant-containing microvesicles from the membrane of aggregating platelets.

One of the responses of platelets to stimulation is activation of intracellular calpain (the Ca(2+)-dependent protease). Previously, we have shown that activation of calpain in platelets is involved in the generation of platelet procoagulant activity. Because procoagulant activity is present on the microvesicles that are shed from activated platelets, in this study we examined whether calpain is involved in the shedding of microvesicles. Platelets were incubated with the physiological agonists collagen or thrombin. The extent of activation of calpain correlated positively with the amount of procoagulant-containing microvesicles that formed, and the shedding of procoagulant-containing microvesicles was inhibited by calpeptin, MDL, and EST (E-64-d), three membrane-penetrating inhibitors of calpain. The protein composition of the microvesicles shed from aggregating platelets was similar to that of microvesicles shed by platelets in which the association of the membrane skeleton with the plasma membrane had been disrupted by incubation of platelets with dibucaine or ionophore A23187. Furthermore, like microvesicles shed from dibucaine- or ionophore A23187-treated platelets, those shed from the aggregating platelets possessed procoagulant activity. These results are consistent with the possibility that activation of calpain in aggregating platelets causes the shedding of procoagulant-containing microvesicles. We suggest that the shedding of microvesicles results from the calpain-induced hydrolysis of the platelet membrane skeleton.

Phosphorus and proton magnetic resonance spectroscopic studies on the relationship between transparency and glucose metabolism in the rabbit lens.

We report the results of a series of nuclear magnetic resonance (NMR) experiments designed to investigate the relationship between particular aspects of glucose metabolism and cataract formation in the rabbit lens. The glucose metabolism of the rabbit lens incubated in TC-199 medium containing 5.5 mM glucose, in glucose-deficient medium, and in modified Earle's medium containing 5.5 mM glucose devoid of NaCl, is examined in conjunction with the assessment of lens transparency. Significant age-dependent differences in the phosphorus metabolite profile and in hexosemonophosphate shunt flux, as measured by NMR, were observed in lenses incubated in TC-199 medium containing 5.5 mM glucose. Incubation in glucose-deficient medium for 8 hr results in significant increases in the levels of inorganic phosphate and phosphomonoesters, and decreases in ATP and L-alpha-glycerolphosphate. These levels regain near-normal values after 24 hr incubation in control medium containing 5.5 mM glucose. By contrast, shunt flux is three times the basal level during the recovery period. Lens clarity, as assessed by slit lamp micrography, was maintained throughout the duration of the experiment. Incubation of adult and juvenile lenses for 18 hr in Earle's medium (pH 7.4 or 9.2) containing 5.5 mM glucose, and no NaCl, results in uniform lenticular opacification within 18 hr and changes in ultrastructure of the epithelial and cortical lens fiber cells. No statistically significant change in the NMR visible phosphorus metabolite profile or intralenticular pH is observed for the adult rabbit lens relative to a lens incubated under control conditions. For the juvenile rabbit lens, small, but statistically significant differences in the levels of dinucleotide and uridinediphosphoglucose were observed. Shunt flux, in contrast, is increased two-fold. These results demonstrate that the NMR visible phosphorus metabolite profile of the lens does not necessarily correlate with transparency, and that hexosemonophosphate shunt activity provides a sensitive measure of prior or current lenticular stress.

On the role of the platelet membrane skeleton in mediating signal transduction. Association of GP IIb-IIIa, pp60c-src, pp62c-yes, and the p21ras GTPase-activating protein with the membrane skeleton.

The platelet plasma membrane is lined with a membrane skeleton composed of short actin filaments, actin-binding protein, spectrin, vinculin, and other unidentified proteins. It is connected to the outside of the cell through association with the cytoplasmic domains of transmembrane receptors. In detergent-lysed platelets, cytoplasmic actin filaments are sedimented by centrifugation at 15,600 x g, but the sedimentation of membrane skeleton fragments requires higher g-forces (100,000 x g). In the present study, we show that the major platelet integrin, glycoprotein (GP) IIb-IIIa, sediments from detergent-lysed platelets at 100,000 x g together with fragments of the membrane skeleton that contain the cytoskeletal proteins spectrin, vinculin, and talin. In addition, this cell fraction contained the tyrosine kinases pp60c-src and pp62c-yes and the p21ras GTPase-activating protein (GAP). After thrombin-induced platelet aggregation mediated by fibrinogen binding to GP IIb-IIIa on adjacent platelets, we detected a redistribution of spectrin, talin, vinculin, pp60c-src, and pp62c-yes to the fraction that sediments at 15,600 x g. The redistribution of these proteins from the high-speed detergent-insoluble fraction to the low-speed fraction correlated with the extent of aggregation and was not detected in aggregation-defective thrombasthenic platelets (which lack the GP IIb-IIIa complex). In addition, many of the proteins phosphorylated on tyrosine in activated platelets were present in detergent-insoluble fractions. These results are consistent with the possibilities that 1) GP IIb-IIIa, pp60c-src, pp62c-yes, and GAP associate with a membrane skeleton fraction that contains spectrin, vinculin, and talin, 2) the association of GP IIb-IIIa with adhesive ligand in a platelet aggregate causes components of the membrane skeleton to undergo altered association with cytoplasmic actin filaments, and 3) many of the proteins phosphorylated on tyrosine residues in activated platelets are components of the cytoskeleton. The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets. Further, GP IIb-IIIa-induced redistribution of components of the membrane skeleton and associated signaling molecules may represent an important step in regulating integrin-induced motile events in platelets.