Clathrin in gastric acid secretory (parietal) cells: biochemical characterization and subcellular localization.

Clathrin from H-K-ATPase-rich membranes derived from the tubulovesicular compartment of rabbit and hog gastric acid secretory (parietal) cells was characterized biochemically, and the subcellular localization of membrane-associated clathrin in parietal cells was characterized by immunofluorescence, electron microscopy, and immunoelectron microscopy. Clathrin from H-K- ATPase-rich membranes was determined to be comprised of conventional clathrin heavy chain and a predominance of clathrin light chain A. Clathrin and adaptors could be induced to polymerize quantitatively in vitro, forming 120-nm-diameter basketlike structures. In digitonin-permeabilized resting parietal cells, the intracellular distribution of immunofluorescently labeled clathrin was suggestive of labeling of the tubulovesicular compartment. Clathrin was also unexpectedly localized to canalicular (apical) membranes, as were alpha-adaptin and dynamin, suggesting that this membrane domain of resting parietal cells is endocytotically active. At the ultrastructural level, clathrin was immunolocalized to canalicular and tubulovesicular membranes. H-K-ATPase was immunolocalized to the same membrane domains as clathrin but did not appear to be enriched at the specific subdomains that were enriched in clathrin. Finally, in immunofluorescently labeled primary cultures of parietal cells, in contrast to the H-K-ATPase, intracellular clathrin was found not to translocate to the apical membrane on secretagogue stimulation. Taken together, these biochemical and morphological data provide a framework for characterizing the role of clathrin in the regulation of membrane trafficking from tubulovesicles and at the canalicular membrane in parietal cells.

Clathrin in mitotic spindles.

Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells.

An immunologically distinct beta-adaptin on tubulovesicles of gastric oxyntic cells.

Clathrin and the gamma-adaptin subunit of the AP-1 clathrin adaptor have been previously identified on H-K-ATPase-rich tubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring. Am. J. Physiol. 274 (Cell Physiol. 43): C1017-C1029]. We further characterized this AP-1 adaptor from rabbit and hog tubulovesicles biochemically and immunologically. Clathrin coat proteins were stripped from purified tubulovesicular membranes and fractionated by hydroxyapatite chromatography. The AP-1 adaptor appears to elute at 200 mM sodium phosphate, based on the presence of proteins in this fraction that are immunoreactive with antibodies against three of the four subunits of this heterotetrameric complex: the gamma-, mu1-, and sigma1-adaptin subunits. Although the putative beta-adaptin subunit in this fraction is not immunoreactive with the anti-beta-adaptin monoclonal antibody (MAb), this beta-adaptin is immunoreactive with polyclonal antibodies (PAbs) directed against the peptide sequence Gly625-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro- Val640 , a region conserved between beta1- and beta2-adaptins that is thought to be involved in the binding of clathrin heavy chain. Immunoprecipitation of the AP-1 adaptor complex from this fraction with anti-gamma-adaptin MAb 100/3 resulted in the coimmunoprecipitation of the beta-adaptin that did not react with the anti-beta-adaptin MAb but did react with the anti-beta-adaptin PAbs. In contrast, immunoprecipitation of the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a beta-adaptin that was recognized by both the anti-beta-adaptin MAb and PAbs. These results suggest that the tubulovesicular AP-1 adaptor complex may be distinct from that found in the trans-Golgi network and may contain an immunologically distinct beta-adaptin. This immunologically distinct beta-adaptin may be diagnostic of apical tubulovesicular endosomes of epithelial cells.

Identification of clathrin and clathrin adaptors on tubulovesicles of gastric acid secretory (oxyntic) cells.

gamma-Adaptin and clathrin heavy chain were identified on tubulovesicles of gastric oxyntic cells with the anti-gamma-adaptin monoclonal antibody (MAb) 100/3 and an anti-clathrin heavy chain MAb (MAb 23), respectively. In Western blots, crude gastric microsomes from rabbit and rat and density gradient-purified, H-K-ATPase-rich microsomes from these same species were immunoreactive for gamma-adaptin and clathrin. In immunofluorescent labeling of isolated rabbit gastric glands, anti-gamma-adaptin and anti-clathrin heavy chain immunoreactivity appeared to be concentrated in oxyntic cells. In primary cultures of rabbit oxyntic cells, the immunocytochemical distribution of gamma-adaptin immunoreactivity was similar to that of the tubulovesicular membrane marker in oxyntic cells, the H-K-ATPase. Further biochemical characterization of the tubulovesicular gamma-adaptin-containing complex suggested that it has a subunit composition that is typical of that for a clathrin adaptor: in addition to the gamma-adaptin subunit, it contains a beta-adaptin subunit and other subunits of apparent molecular masses of 50 kDa and 19 kDa. From solubilized gastric microsomes from rabbit, gamma-adaptin could be copurified with the major cargo protein of tubulovesicles, the H-K-ATPase. Thus this tubulovesicular coat may bind directly to the H-K-ATPase and may thereby mediate the regulated trafficking of the H-K-ATPase at the apical membrane of the oxyntic cell during the gastric acid secretory cycle. Given the similarities of the regulated trafficking of the H-K-ATPase with recycling of cargo through the apical recycling endosome of many epithelial cells, we propose that tubulovesicular clathrin and adaptors may regulate some part of an apical recycling pathway in other epithelial cells.

Blood pressure lowering by pioglitazone. Evidence for a direct vascular effect.

To examine potential mechanisms for the blood pressure-lowering action of the thiazolidinedione compound, pioglitazone (PIO), we studied the effects of the drug on blood pressure and insulin action in vivo and on vascular tissue in vitro. In vivo, PIO lowered blood pressure in fructose-fed and chow-fed rats to an extent that could not be explained by alterations in fasting plasma insulin or free magnesium concentrations or by alterations in whole-body insulin sensitivity. In vitro, PIO caused significant blunting of the contractile responses of aortic rings to NE, arginine vasopressin (AVP), and potassium chloride; the blunting of responses to NE was maintained after removal of the endothelium. To assess the potential importance of extracellular calcium to the vasodepressor effect of PIO, we measured contractile responses to NE in the absence of calcium, and then after acute restoration of calcium in the presence of NE. PIO had no effect on the contractile response in the absence of calcium. By contrast, PIO blunted by 42% the contractile response that occurred when the extracellular calcium supply was acutely restored in the presence of NE, suggesting that the blunting was mediated by blockade of calcium uptake by vascular smooth muscle. Such an effect was confirmed in cultured a7r5 vascular smooth muscle cells, which exhibited a brisk increase in intracellular calcium in response to AVP that was blocked by PIO in a dose-dependent fashion. Our data indicate that PIO has a direct vascular effect that appears to be mediated at least in part by inhibition of agonist-mediated calcium uptake by vascular smooth muscle. The direct vascular effect may contribute to the blood pressure-lowering actions of PIO in vivo, because that effect could not be explained by alterations in whole-body insulin sensitivity.

Endothelial-dependent vascular effects of insulin and insulin-like growth factor I in the perfused rat mesenteric artery and aortic ring.

Insulin and insulin-like growth factor I (IGF-I) exhibit vasoactivity. To examine the role of the endothelium in mediating the vascular responses to insulin and IGF-I, we exposed both isolated intact rat mesenteric arteries and rat aortic rings to these growth factors in the presence and absence of endothelium. Perfusion of rat mesenteric arteries with insulin, IGF-I, or IGF-II resulted in the potentiation of arginine vasopressin (AVP)-induced vasoconstriction. Of these growth factors, IGF-I was the most potent, with a significant effect at 0.6 nM and maximal effects at 6.0 nM, followed by IGF-II and insulin. Endothelial denudation or addition of cycloheximide prevented the growth-factor effects. Tissue cGMP levels in the mesenteric artery were minimally affected by growth factors. Insulin and IGF-I vascular effects were not inhibited by BQ123, an endothelin (ET) antagonist that blocked ET-1 enhancement of AVP response. Perfusion of mesenteric arteries with IGF-I for 1 h did not alter vessel ET-1 or ET-1 mRNA contents. Addition of indomethacin markedly inhibited the IGF-I effect on AVP contraction. Thus, the mesenteric vascular effect of insulin and IGF-I is not associated with ET-1 release but appears to link to an increased release of an endothelial-derived contracting factor or the decreased production of an endothelial-derived relaxing factor from the cyclooxygenase pathway. In contrast to their action in the mesenteric artery, insulin (exceeding 100 nM) and IGF-I (1-30 nM) attenuated AVP- and norepinephrine-induced contraction in rat aortic rings. Endothelial-denudation abolished this effect.(ABSTRACT TRUNCATED AT 250 WORDS)