Altered hepatic transport of immunoglobulin A in mice lacking the J chain.

We have created J chain knockout mice to define the physiologic role of the J chain in immunoglobulin synthesis and transport. The J chain is covalently associated with pentameric immunoglobulin (Ig) M and dimeric IgA and is also expressed in most IgG-secreting cells. J chain-deficient mice have normal serum IgM and IgG levels but markedly elevated serum IgA. Although polymeric IgA was present in the mutant mice, a larger proportion of their serum IgA was monomeric than was found in wild-type mouse serum. Bile and fecal IgA levels were decreased in J chain-deficient mice compared with wild-type mice, suggesting inefficient transport of J chain-deficient IgA by hepatic polymeric immunoglobulin receptors (pIgR). The pIgR-mediated transport of serum-derived IgA from wild-type and mutant mice was assessed in Madin-Darby canine kidney (MDCK) cells transfected with the pIgR. These studies revealed selective transport by pIgR-expressing MDCK cells of wild-type IgA but not J chain-deficient IgA. We conclude that although the J chain is not required for IgA dimerization, it does affect the efficiency of polymerization or have a role in maintaining IgA dimer stability. Furthermore, the J chain is essential for efficient hepatic pIgR transport of IgA.

Turning on ARF: the Sec7 family of guanine-nucleotide-exchange factors.

ARF proteins are important regulators of membrane dynamics and protein transport within the eukaryotic cell. The Sec7 domain is approximately 200 amino acids in size and stimulates guanine-nucleotide exchange on members of the ARF class of small GTPases. The members of one subclass of Sec7-domain proteins are direct targets of the secretion-inhibiting drug brefeldin A, which blocks the exchange reaction by trapping a reaction intermediate in an inactive, abortive complex. A separate subclass of Sec7-domain proteins is involved in signal transduction and possess a domain that mediates membrane binding in response to extracellular signals.

Gs alpha stimulates transcytosis and apical secretion in MDCK cells through cAMP and protein kinase A.

Recent evidence suggests a role for heterotrimeric G proteins in vesicular transport. Cholera toxin, which activates Gs alpha by ADP-ribosylation, has been reported to stimulate both apical secretion (Pimplikar, S.W., and K. Simons. 1993. Nature (Lond.). 352:456-458) and apically directed transcytosis (Bomsel, M., and K.E. Mostov. 1993. J. Biol. Chem. 268:25824-25835) in MDCK cells, via a cAMP-independent mechanism. Here, we demonstrate that apical secretion and apically directed transcytosis are significantly stimulated by agents that elevate cellular cAMP. Forskolin, which activates adenylyl cyclase directly, and 8BrcAMP augment both transport processes in MDCK cells. The increase is not limited to receptor-mediated transport (polymeric Ig receptor), since transcytosis of ricin, a galactose-binding lectin, is similarly stimulated. The effects of elevated cellular cAMP on apical secretion and transcytosis are apparently mediated via protein kinase A (PKA), as they are inhibited by H-89, a selective PKA inhibitor. Experiments employing a 17 degrees C temperature block indicate that cAMP/PKA acts at a late, possibly rate-limiting stage in the transcytotic pathway, after translocation of internalized markers into the apical cytoplasm. However, no significant stimulus of apical recycling was observed in the presence of FSK, suggesting that cAMP/PKA either affects transcytosis at a level proximal to apical early endosomes and/or specifically increases the efficiency by which transcytosing molecules are delivered to the apical plasma membrane. Finally, we overexpressed wild-type Gs alpha and a mutant, Q227L, which constitutively activates adenylyl cyclase, in MDCK cells. Although Q227L increased transcytosis more than wild-type Gs alpha, neither construct was as effective as FSK in stimulating transcytosis, arguing against a significant role of Gs alpha in transcytosis independent of cAMP and PKA.

Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D.

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.

Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis.

The endosomal compartment of polarized epithelial cells is a major crossroads for membrane traffic. Proteins entering this compartment from the cell surface are sorted for transport to one of several destinations: recycling to the original cell surface, targeting to lysosomes for degradation, or transcytosis to the opposite surface. The polymeric immunoglobulin receptor (pIgR), which is normally transcytosed from the basolateral to the apical surface, was used as a model to dissect the signals that mediate this sorting event. When exogenous receptor was expressed in Madin-Darby Canine Kidney (MDCK) cells, it was shown that phosphorylation of pIgR at the serine residue at position 664 is required for efficient transcytosis. Replacement of this serine with alanine generated a receptor that is transcytosed only slowly, and appears to be recycled. Conversely, substitution with aspartic acid (which mimics the negative charge of the phosphate group) results in rapid transcytosis. It was concluded that phosphorylation is the signal that directs the pIgR from the endosome into the transcytotic pathway.

Regulation of ARNO nucleotide exchange by a PH domain electrostatic switch.

ARNO is a member of a family of guanine nucleotide exchange factors that activate small GTPases called ADP-ribosylation factors (ARFs) [1] [2] [3], which regulate vesicular trafficking and, in one case (ARF6), also regulate cortical actin structure [4]. ARNO is located at the plasma membrane, and in the presence of activated protein kinase C (PKC) can induce cortical actin rearrangements reminiscent of those produced by active ARF6 [5] [6] [7] [8]. High-affinity binding of ARNO to membranes, which is required for exchange activity, is mediated cooperatively by a pleckstrin homology (PH) domain and an adjacent carboxy-terminal polybasic domain [3] [9]. ARNO is phosphorylated in vivo by PKC on a single serine residue, S392, located within the carboxy-terminal polybasic domain. Mutation of S392 to alanine does not prevent ARNO-mediated actin rearrangements, suggesting that phosphorylation does not lead to ARNO activation [6]. Here, we report that phosphorylation negatively regulates ARNO exchange activity through a 'PH domain electrostatic switch'. Introduction of a negatively charged phosphate into the polybasic domain reduced interaction of ARNO with membranes both in vitro and in vivo, and inhibited exchange in vitro. This regulated membrane association is similar to the myristoyl electrostatic switch that controls membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) [10], but to our knowledge is the first demonstration of an electrostatic switch regulating the membrane interaction of a protein containing a PH domain. This mechanism allows regulation of ARNO lipid binding and exchange activity at two levels, phosphoinositide-dependent recruitment and PKC-dependent displacement from the membrane.

Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO.

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.

Association of Rab25 and Rab11a with the apical recycling system of polarized Madin-Darby canine kidney cells.

Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.

In vitro phosphorylation of caveolin-rich membrane domains: identification of an associated serine kinase activity as a casein kinase II-like enzyme.

Caveolae are flask-shaped micro-invaginations associated with the plasma membrane of a wide variety of cell types. Caveolin, an integral membrane component of caveolae, was first identified as the major phosphoprotein whose phosphorylation was elevated in v-Src transformed cells. As both v-Src transformation and elevated caveolin phosphorylation were dependent on membrane attachment of v-Src, it has been suggested that caveolin is a critical target in v-Src transformation. Although an increase in tyrosine phosphorylation of caveolin was evident, the increase in caveolin phosphorylation was predominantly on serine residues. In accordance with these in vivo observations, isolated caveolin-rich membrane domains undergo phosphorylation in vitro predominantly on serine and contain an unidentified serine kinase activity. Here, we have identified this serine kinase activity as a casein kinase II-like enzyme, since the phosphorylation of caveolin-rich membrane domains is stimulated and inhibited by known effectors of casein kinase II (poly-L-lysine, endogenous polyamines, and a casein kinase II inhibitor peptide), but is unaffected by modulators of other known kinases. In support of these observations, caveolin contains a consensus sequence for casein kinase II phosphorylation in its cytoplasmic N-terminal domain (Ser-88). A peptide containing this sequence inhibits the in vitro phosphorylation of caveolin-rich membrane domains, while many other peptides derived from the N-terminal domain of caveolin do not affect phosphorylation. Caveolin-rich membrane domains were also a substrate for exogenously added purified casein kinase II, but not casein kinase I. Finally, immunoblotting of these domains with an antibody directed against the alpha and alpha' subunits of casein kinase II reveals two bands with apparent molecular weights consistent with the known molecular weights of the alpha and alpha' subunits of casein kinase II. As casein kinase II appears to play a role in mitogenic signalling events and casein kinase II activators (endogenous polyamines) are required for v-Src transformation, our results may have implications for understanding the mechanism of v-Src oncogenesis.

Bronchial hyperreactivity and cough due to angiotensin-converting enzyme inhibitors.

Seventeen patients using angiotensin-converting enzyme (ACE) inhibitors for hypertension were evaluated with baseline spirometry followed by determination of bronchial reactivity by challenge with methacholine. There were nine coughers and eight noncoughers in the study. Among the nine coughers, eight demonstrated bronchial hyperreactivity. Conversely, none of the noncoughers disclosed bronchial hyperreactivity. Eight of the nine coughers were rechallenged two to six months following cessation of ACE inhibitor therapy. Six of these eight showed persistent bronchial hyperreactivity. We conclude that cough is associated with the use of ACE inhibitors in patients with underlying bronchial hyperreactivity. The findings indicate caution in administration of ACE inhibitors in asthmatic patients and those with known bronchial hyperreactivity.

Hand dexterity in hospital personnel with multiple needlestick injuries.

OBJECTIVE: To test the hypothesis that multiple needlestick injuries in hospital employees may be due to intrinsic deficits in hand dexterity. DESIGN: A case-control study comparing employees with multiple reported needlestick injuries to those with none. Hand dexterity was tested using the Purdue Pegboard Test, a standardized validated test of hand dexterity. SETTING: A 300-bed, acute care teaching hospital. PARTICIPANTS: Fifteen hospital employees who sustained four or more injuries were compared to 19 controls. RESULTS: No differences were detected in hand dexterity between the case and control groups. CONCLUSIONS: While employees with multiple needlestick injuries accounted for 85% of reported injuries, underlying factors responsible for injuries in this high-risk subgroup do not include measurable deficits in hand dexterity.

US family physicians' experiences with practice guidelines.

BACKGROUND: Practice guidelines were developed to improve medical outcomes and cost-effectiveness. The experiences of family physicians, who may need to use multiple guidelines in their practices, are crucial for effective development and implementation of practice guidelines. We surveyed a national sample of US family physicians about factors that affect their adoption and use of practice guidelines. METHODS: We mailed a structured survey to a national random sample of 400 family physician members of the American Academy of Family Physicians. RESULTS: The response rate was 51%. Most respondents (69%) reported a positive attitude about practice guidelines, but only 44% reported using any guidelines. More younger physicians thought that guidelines could be useful tools. Most preferred guidelines that could be modified (87%) and that were no longer than two pages. Only 27% of respondents knew where to locate a guideline on a particular topic. Forty-three percent of respondents reported that it would be useful if guidelines were a component of an electronic medical record. CONCLUSIONS: If guidelines are to be used by practicing family physicians, a generalist perspective needs to be considered in future guideline development and implementation. Younger physicians had more positive attitudes toward guidelines.

Lessons from the Soviet health care system.

The United States and the former Soviet Union have historically organized health care delivery systems according to totally different paradigms. These two divergent approaches have constituted a kind of natural experiment. At the present time, our systems may be becoming more alike, with the former Soviet system decentralizing and even experimenting with forms of medical insurance. Our system, on the other hand, has become much more regulated and, if some have their way, would become increasingly monopsonistic. At this critical point, it may be useful to learn from each other's experiences as we plan for the future.

The outgrowth of parietal endoderm from mouse teratocarcinoma stem-cell embryoid bodies.

Teratocarcinoma stem cells can be used to study certain events occurring during early mouse embryogenesis. We report that the outgrowth of parietal endoderm from teratocarcinoma stem-cell embryoid bodies in vitro is analogous to the same process in vivo in terms of the spatial distribution of endoderm types: only parietal endoderm migrates away from the aggregate, whereas visceral endoderm remains associated with the embryoid body. The outgrowths generated on a substrate of type-I collagen from PSA-1 and retinoic-acid-treated F 9 embryoid bodies were found to be comparable, even though these aggregates express different endoderm types. We demonstrated that retinoic-acid-treated F 9 embryoid bodies that contain essentially only visceral endoderm in suspension culture can nonetheless generate parietal-endoderm outgrowth when plated on type-I collagen, suggesting that substrate interaction plays an important role in inducing parietal-endoderm differentiation. These data indicate the usefulness and relevance of studying endoderm differentiation and outgrowth in vitro employing the teratocarcinoma model system.