James D. Jamieson

Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes

Madin Darby Canine Kidney (MDCK) cells grown on polycarbonate filters in a two-chamber culture system were used to study the postsynthetic sorting of the alpha-subunit of the (Na+,K+)ATPase, an important native protein of the MDCK cell basolateral plasmalemmal domains. The N-azidobenzoyl derivative of ouabain (NAB-ouabain) and anti-ouabain antibodies were used in pulse labeling experiments to monitor the arrival of newly synthesized molecules of (Na+,K+)ATPase at the apical and basolateral cell surfaces. The results show that newly synthesized alpha-subunits bind NAB-ouabain and become substrates for immunoprecipitation only when this compound is present in the basolateral chamber. No more than 10% of the (Na+,K+)ATPase synthesized during the pulse period could appear at the apical surface without being detected by our assay. Thus, sorting of this native protein is effected intracellularly prior to its direct insertion into the basolateral plasmalemmal domain. Passage through an acidic compartment is not required for proper sorting.

Rab3D Is Not Required for Exocrine Exocytosis but for Maintenance of Normally Sized Secretory Granules

ABSTRACT Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.

Chapter 24 Sequential Dissociation of the Exocrine Pancreas into Lobules, Acini, and Individual Cells

Publisher Summary The chapter outlines the procedure for the sequential dissociation of the exocrine pancreas into lobules, acini, and individual cells. Dispersion of the pancreas into isolated single cells involves sequential enzymic digestion of stromal collagen and basement membrane, Ca2+ chelation to disrupt cell-cell junctions (that is, desmosomes and tight junctions), and mechanical shearing to complete the separation of gap and tight junctions. Filtration and washing of the tissue digest yield a purified preparation free of cell debris, collagenase, and subcellular organelles liberated in the final steps of the procedure. The protocol for tissue dissociation described allows the investigator to control the extent of dissociation of pancreatic exocrine tissue into minilobules, single acini, and separated cells, and to analyze how the progression of dissociation affects cell structure and function. Treatment of the tissue with purified collagenase and with mild mechanical shearing forces was found to be sufficient to dissociate the tissue into minilobules and single acini, whereas an additional step of Ca2+ chelation was found to be essential to break down the junctional elements between cells in order to obtain separated cell.

Sodium Gradient, Smooth Muscle Tone, and Blood Pressure Regulation

Rat colon strips respond to an acute reduction of sodium concentration in the medium (Nao) by an immediate increase in tension, followed by relaxation to the basal tension as the tissue equilibrates. Following equilibration in low Nao the responsiveness to drug-induced contraction is increased, while in high Nao it is decreased. A contraction cycle, variously induced, can be aborted by the acute addition of sodium to the medium. The applicability of these findings to vascular smooth muscle in the whole animal can be demonstrated by appropriate acute sodium infusions. The effects in all cases can be referred to sodium, although modified by the anionic component of the salts used. The results are considered to support the view that smooth muscle tension is governed in part by the concentration gradient Nao/Nai and the theoretic implication of this for chronic hypertensive and hypotensive states is developed.

Development of secretagogue response in rat pancreatic acinar cells.

Abstract Two to 3 days prior to birth, acinar cells of the rat pancreas acquire morphologic and biochemical characteristics of the adult gland. To determine if differentiation of the secretory apparatus coincides temporally with the capacity of the cell to respond to secretory stimuli, lobules of embryonic, neonatal, and adult rat pancreas were compared for their ability to respond to secretagogues presumed to act directly via hormone receptors [caerulein and carbamylcholine (carbachol)] or indirectly (cyclic nucleotide analogs and the Ca 2+ ionophore A23187). Of all agents tested, only dibutyryl cAMP elicited discharge of secretory proteins at day 20 in utero and preceded hormone stimulation by 1 day. A23187 elicited discharge by Day 21 in utero ; its action was near adult levels in contrast to hormonal stimuli whose effect was maximal only at birth. All secretagogues required Ca 2+ and energy to induce discharge. Pulse-chase autoradiography of lobules from Day 20 embryonic glands indicated that the acinar cells were capable of transporting [ 3 H]leucine-labeled proteins to zymogen granules at rates roughly equivalent to those in adult glands. SDS gel electrophoretograms confirmed that the bulk of 14 C-amino acid incorporation into proteins at a given age was primarily into exportable proteins. The results indicate that acinar cells synthesize and package secretory proteins into zymogen granules about 2 days before they are capable of responding to hormonal stimuli and to intracellular effectors.

Guinea pig pancreatic acini prepared with purified collagenase

Abstract Dispersed guinea pig pancreatic acinar cells have been used to investigate several aspects of stimulus-secretion coupling but possess the disadvantage that they are less sensitive and less responsive to secretagogues than in vitro preparations of intact pancreatic tissue (lobules). To overcome the poor responsiveness of isolated acinar cells, we have developed a new procedure for preparing dispersed, intact pancreatic acini whose sensitivity to secretagogues and morphological characteristics are similar to those of pancreatic lobules. Dispersed acini can be manipulated as suspensions of cells and full access of macromolecular probes to apical and basolateral plasmalemmal domains is obtained. Acini were prepared in good yields (~70% on a DNA basis) using only purified collagenase and mild mechanical shear in medium containing 2.0 mM Ca 2+ . Morphologically, acinar cells in the preparations retained intact junctional complexes, asymmetrical distribution of intramembranous particles between apical and basolateral plasmalemmal domains, and polarized distribution of intracellular organelles as found in intact pancreas. Dose-response curves of acini and mechanically prepared lobules to caerulein, carbachol, and bombesin were similar though acini were more sensitive to the C-terminal octapeptide of cholecystokinin. Net stimulated secretory protein discharge was ~36% over 2 h. Crude collagenase was purified for use in preparation of acini by Sephadex G-75 column chromatography which resolved collagenase from clostripain and a non-sulfhydryl-requiring protease. The purified collagenase contained at least four proteins with molecular weights between 85 000 and 110 000. Collagenase with 0.9 units of protease per unit of collagenase yielded unresponsive acini. Acini incubated with crude collagenase, chymotrypsin, or the non-sulfhydryl-requiring protease showed depressed secretory response to caerulein. Freeze-fracture electron microscopy of protease-treated acini indicated that the intramembranous particles aggregated and that many of the tight junctions had undergone a proliferation of non-cross-linked sealing strands which extended far down the basolateral plasma membrane and encircled gap junctions. Acini incubated with purified collagenase or with a clostripain-containing fraction from the Sephadex G-75 column appeared unaltered. This procedure produces acini which are morphologically and biochemically similar to the in situ pancreas and overcomes the poor response to secretagogues by isolated pancreatic acinar cells.

Regional differences in lectin binding to colonic epithelium by fluorescent and electron microscopy.

The distribution of glycosubstances in colonic epithelium using lectins that were rhodaminated for fluorescent microscopy or coupled to colloidal gold for electron microscopy has been studied. Findings, based on lectin binding patterns, indicate that the right, transverse, and left colon of the guinea pig differ in their content of glycoconjugates within goblet cells and along the brush border. Local variations of labeling were also observed within goblet cells between the upper and lower portions of crypts. Throughout the colon, corresponding to a region of supranuclear fluorescence, Ricin II-colloidal gold labeled the Golgi complex of both enterocytes and goblet cells. Ricin II-colloidal gold also labeled small (congruent to 800 A) vesicles in the apical portion of enterocytes in all colonic segments. Microvilli were labeled by Ricin II-colloidal gold in the right and transverse colon, a finding that correlated with the observed adhesion of bacteria in those segments.

Cell surface properties of normal, differentiating, and neoplastic pancreatic acinar cells

Acinar, centroacinar, and endocrine cells of the adult rat pancreas each exhibit distinctive cell‐surface glycoconjugate patterns as detected by binding of a battery of lectin‐ferritin conjugates. Acquisition of these unique glycoconjugate patterns appears to be developmentally regulated, as studies on embryonic rat pancreases at days 15, 17, and 19 of gestation indicate. Further, the three cell types appear to arise from a common stem cell(s) with surface glycoconjugate properties similar to those of the adult centroacinar cell. Studies on the cell‐surface properties of a rat acinar cell tumor20 indicate that the neoplastic acinar cells are likely to be arrested at a developmental stage equivalent to acinar cells of the day 19 embryonic pancreas and are characterized by absence of detectable basal lamina. We hypothesize that pancreatic cancers may arise from the equivalent of the undifferentiated embryonic stem cell(s) and that the morphologic features of the tumor depend on the extent of cell differentiation, including expression of cell‐surface glycoproteins and extracellular matrix, prior to neoplastic proliferation.

Immunocytochemical localization of BiP to the rough endoplasmic reticulum: evidence for protein sorting by selective retention.

Immunoglobulin heavy chain binding protein (BiP) (also known as GRP 78) is a protein of the endoplasmic reticulum (ER) which has been shown to be involved in post-translational processing of nascent membrane and secretory proteins. To determine BiPs location in the exocytic pathway, we localized BiP at the electron microscopic level in mouse myeloma cell lines by immunoperoxidase cytochemistry. BiP was found to be present within the cisternal spaces of the RER and nuclear envelope but was not detected in the cisternae of the Golgi complex. BiP reaction product was also found within transitional elements of the RER but was absent from smooth-surfaced vesicles found between the ER and the Golgi complex. Immunoperoxidase staining of BiP was reduced or absent in regions of a smooth ER membrane system in myelomas that contained endogenous murine retrovirus A particles. All compartments of the exocytic pathway, including the virus-containing smooth ER, stained for IgG, a secretory protein. These observations suggest that BiP is selectively retained in the cisternae of the ER and is not free to enter Golgi-directed transport vesicles. These studies suggest that BiPs subcellular localization may occur by selective interaction with component(s) of the ER.

Purification and use of limulin: a sialic acid-specific lectin.

A simple and rapid method for the isolation of the sialic acid-specific lectin, Limulus polyphemus hemagglutinin (LPA), from the hemolymph of Limulus polyphemus is described. Declotted hemolymph is adsorbed to an affinity chromatographic column consisting of hog gastric mucin glycopeptides coupled to agarose and LPA is eluted in a single step with a Ca2+-free buffer, giving an apparent purification of approximately 25,000-fold. The eluted material is homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing and consists of identical subunits each of 29,000 daltons. Hemagglutination inhibition studies with horse red blood cells indicate specificity of LPA for N-acetyl- and N-glycolylneuraminic acid; binding is Ca2+ -dependent and abolished by neuraminidase treatment. LPA was covalently coupled to rhodamine and to horseradish peroxidase for use in detection of sialoglycoconjugates on cells and tissues by light and electron microscopy. Examples of the use of LPA for detection of sialoglycoconjugates in rat renal tubules and glomeruli, blood vessels in rat pancreas, and on horse red blood cells are shown. The procedures described here should prove useful as a cytochemical probe for detection of sialoglycoconjugates in a variety of systems. An accompanying article utilizes these probes for the detection of sialoglycoconjugates on the plasmalemma of adult and differentiating rat pancreatic acinar cells.