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Distinctive functions of membrane type 1 matrix-metalloprotease (MT1-MMP or MMP-14) in lung and submandibular gland development are independent of its role in pro-MMP-2 activation.

Membrane type 1-matrix metalloprotease (MT1-MMP or MMP-14) is a major activator of pro-MMP-2 and is essential for skeletal development. We show here that it is required for branching morphogenesis of the submandibular gland but not the lung. Instead, in the lung, it is essential for postnatal development of alveolar septae. Lung development in Mmp14-/- mice is arrested at the prealveolar stage with compensatory hyperinflation of immature saccules. Mmp2-/- mice lacked comparable defects in the lung and submandibular gland, suggesting that MT1-MMP acts via mechanisms independent of pro-MMP-2 activation. Since the developmental defects in the lung are first manifest around the time of initial vascularization (E16.5), we investigated the behavior of pulmonary endothelial cells from Mmp14+/+ and Mmp14-/- mice. Endothelial cells from lungs of 1-week-old Mmp14-/- mice show reduced migration and formation of three-dimensional structures on Matrigel. Since pulmonary septal development requires capillary growth, the underlying mechanism of pulmonary hypoplasia in Mmp14-/- mice may be defective angiogenesis, supporting a model in which angiogenesis is a critical rate-limiting step for acquisition of pulmonary parenchymal mass.

Animals↗

Nitric oxide in the control of submandibular gland function in the anaesthetized ferret.

Stimulation of parasympathetic innervation of the submandibular gland (2 or 20 Hz continuously or 20 Hz for 1 s at 10 s intervals), in the ferret, produced secretion of fluid and protein and a fall in vascular resistance. The responses following the administration of N omega-nitro-L-arginine methyl ester (L-NAME; 2 mmol kg-1 i.a.) to block the synthesis of nitric oxide (NO) were reduced, and the persisting responses were abolished (at 2 Hz continuously and 20 Hz intermittently) or further reduced (at 20 Hz continuously) by the additional administration of atropine. The output of vasoactive intestinal peptide (VIP) from the gland was not affected. Neither the secretory nor the vascular response to intra-arterial infusions of acetylcholine (1.25 nmol kg-1) was affected by L-NAME, whereas the vascular responses to both VIP (10 pmol kg-1) and pituitary adenylate cyclase-activating peptide (1-38) (PACAP) (0.5 pmol kg-1) were reduced thereby. Neither peptide evoked a fluid secretion per se. However, when infused during parasympathetic stimulation of saliva secretion, VIP increased both flow rate and the output of protein. These effects of VIP were abolished by L-NAME. The experiments were performed in the presence of sodium nitroprusside at doses (4-8 nmol min-1 kg-1 i.v.) aimed to counterbalance the systemic effects of L-NAME. The results show that, in the ferret, parasympathetic nerve activity increases submandibular blood flow, and elicits the flow of saliva and output of protein by mechanisms that involve in situ generation of NO, upon which the effects of VIP and PACAP but not acetylcholine are largely dependent.

Animals↗

Immunocytochemical localization of epidermal growth factor in mouse submandibular gland.

The cellular and subcellular localization of epidermal growth factor in the submandibular glands of male and female adult mice was established by immunoperoxidase techniques. In light microscopic preparations epidermal growth factor was found exclusively in the granular convoluted tubules of the gland. The intensity of staining for epidermal growth factor varied from cell to cell, and some cells apparently were negative. The pattern of staining was similar in the glands of male and female mice; however, the granular convoluted tubules are androgen-responsive, and thus more extensive and composed of larger cells in males. In thin sections epidermal growth factor was most heavily concentrated in the secretion granules of the granular convoluted tubule cells. Within a given cell there was variation in intensity of staining of individual secretion granules, with some granules appearing minimally reactive or negative. The only other cell component with deposits of reaction product was the ribosomes.

Animals↗

Studies on creatine kinase in rat submandibular gland.

1. Creatine kinase activities in cytosol and mitochondrial fractions of rat submandibular gland were separated clearly by DEAE cellulose column chromatography and by electrophoresis on cellulose acetate strips. 2. Creatine kinase in cytosol fraction was brain type. 3. A portion of mitochondrial creatine kinase was tightly bound to mitochondria, and was solubilized with Triton X-100. 4. Mitochondrial creatine kinase was separated into 2 portions by DEAE cellulose column chromatography, but not by electrophoresis and gel filtration. 5. Kinetic analysis indicated mitochondrial creatine kinase was directed for the formation of creatine phosphate.

Animals↗

Characterization of the muscarinic cholinergic receptor in the opossum (Didelphis virginiana, Kerr) submandibular gland: differences in receptor density and subtype compared with higher mammalian species.

1. Kinetic, saturation and inhibition radioligand binding experiments with [3H]-N-methylscopolamine and [3H]quinuclidinyl benzilate were used to characterize the muscarinic cholinergic receptor in opossum (Didelphis virginiana, Kerr) submandibular salivary gland membranes. 2. The receptor density in opossum submandibular gland was found to be more than 3-fold higher than in rat, and 22-fold higher than in human, submandibular glands. 3. Inhibitor equilibrium dissociation constants for the antagonists pirenzepine, dicyclomine, atropine, N-methylscopolamine and AF-DX 116 revealed that the muscarinic receptor present in opossum submandibular gland appears to be the M1 subtype rather than the M3 subtype found in human and rat.

Animals↗

A study of vasoactive intestinal peptide and acetylcholine as possible mediators of vasodilatation in the cat submandibular gland.

Vasoactive intestinal polypeptide (VIP) is a powerful vasodilator agent in the submandibular gland of the cat, and its effect can be reduced by avian pancreatic polypeptide (APP), or by desensitization of the gland's blood vessels to VIP. However, the vasodilatation caused by parasympathetic nerve stimulation is not reduced by either of these means. We conclude, therefore, that VIP is unlikely to be a major mediator of this atropine-resistant vasodilatation. Experiments with a potentiator of acetylcholine, eserine, and with a depleter, suggest that acetylcholine plays some role in this vasodilatation, but it too does not appear to be the major physiological mechanism. Substance P and ATP were neither potent nor consistent vasodilators and are unlikely mediators.

Acetylcholine↗

Effect of pyridoxal 5'-phosphate on Ca2+-stimulated adenosine triphosphatase activity in microsomes of rat submandibular gland in vitro.

1. The Ca2+-ATPase activity in microsomes of rat submandibular gland was inhibited by pyridoxal 5'-phosphate in vitro. 2. The dissociation constant of the enzyme-pyridoxal 5'-phosphate complex was estimated to be 6.5 mM. 3. The inhibition of pyridoxal 5'-phosphate for both ATP and Ca2+ was competitive. 4. The order of inhibitory effectiveness of pyridoxal 5'-phosphate analogs was pyridoxal 5'-phosphate greater than pyridoxal HCl greater than pyridoxamine 5'-phosphate greater than pyridoxamine HCl. 5. The enzyme-pyridoxal 5'-phosphate complex was nonreducible with sodium borohydride.

Adenosine Triphosphate↗

Regulation of trypsin-like esteroprotease synthesis by 5 alpha-dihydrotestosterone and triiodothyronine in mouse submandibular gland.

The hormonal regulation of trypsin-like esteroprotease synthesis in mouse submandibular gland was studied at the isozyme level. Antiserum to a mixture of two purified esteroproteases precipitated all the esteroproteases in a crude extract of this gland. Measurement of incorporation of [3H]leucine showed that total esteroprotease synthesis was stimulated by both 5 alpha-dihydrotestosterone and triiodothyronine and that the two hormones had synergistic effects. The observed correlation between the increases of synthetic rate and specific activity of this enzyme suggests that the enzyme level is regulated mainly by the rate of enzyme synthesis. Newly synthesized esteroprotease-antibody complexes gave four peaks of radioactivity with esteroprotease activity and one peak without enzyme activity on isoelectric focusing in acrylamide gel containing 8 M urea. The radioactivities of these five peaks were increased similarly by the two hormones separately or in a combination. These results suggest that the actions of androgens and thyroid homrones in esteroprotease synthesis are indistinguishable at the isozyme level.

Animals↗

T-kininogenase activity of the rat submandibular gland is predominantly due to the kallikrein-like serine protease antigen gamma.

T-kininogen, the major kininogen in rat plasma, releases Ile-Ser-bradykinin (T-kinin) when incubated with trypsin, but is not a substrate for tissue kallikrein. Enzymes able to release T-kinins from T-kininogen have been found in the rat submandibular gland, but precise identification of these enzymes and their possible relationship to kallikrein-like enzymes has not been established. We studied T-kininogenase activity in fractionated submandibular gland homogenate. The main T-kininogen catalytic enzyme was purified and characterized, and found to be identical to antigen gamma, a kallikrein-like enzyme which we have previously characterized. Of other identified kallikrein-like enzymes only tonin showed weak T-kininogenase activity, which was about 0.25% of that of antigen gamma. No other T-kininogen catalytic enzymes were observed. Antigen gamma released a kinin which was identified as T-kinin by reverse-phase h.p.l.c. The T-kininogenase activity of antigen gamma had a Km of 29 +/- 4 microM and a kcat/Km of 140 M-1.s-1, and was comparable with its high and low molecular mass-kininogenase activity (7.4 and 10 micrograms of kinin/h per mg respectively). In contrast, tissue kallikrein released 0.2 and 42,200 micrograms of kinin/h per mg respectively. Thus antigen gamma is a weak kininogenase. The isoelectric point of antigen gamma, but not its molecular mass, differed from that of other kallikrein-like enzymes. Isoelectrofocusing in flat-bed gels combined with immunostaining was therefore a convenient method for identification. The kallikrein-like nature of antigen gamma was demonstrated by its immunological similarity to tissue kallikrein and tonin and by 91% and 87% amino acid sequence similarity with tonin and kallikrein respectively (67 amino acids sequenced). Complete identity was also not observed with other sequenced kallikrein genes, mRNAs or proteins.

Amino Acid Sequence↗

Accumulation of c-fos mRNA in slices of mouse submandibular gland incubated in vitro.

Incubation of slices or isolated lobules of murine submandibular gland at 37 degrees C in physiologic solutions or in tissue culture media, or dissociation of the cells by collagenase-hyaluronidase treatment, increased the steady-state level of c-fos mRNA without any additional stimulus. This activation of c-fos expression required the presence of Na+ and K+ but not extracellular Ca2+. It was augmented by depolarizing concentrations of K+ and by veratridine, and inhibited by high concentrations of amiloride. Alterations in membrane permeability and in ion fluxes and/or perturbation in membrane phospholipids may play a role in this transitional activation of the c-fos gene expression in incubated tissue slices in which the cells are not viable and undergo a necrobiotic process.

Animals↗

Possible role of the submandibular gland in the development of obesity in mice.

The possible role of submandibular glands (SMG) in the development of obesity was studied in two types of genetically obese mice, ob/ob and yellow Ay: Obesity is caused by hyperplasia followed by hypertrophy in ob/ob mice and mainly by hypertrophy in Ay mice. The histological features of SMGs exhibited clear sexual dimorphism in both mice similar to lean controls. The SMGs of ob/ob mice was smaller in size and had smaller granular convoluted tubular portions than lean controls, while the SMGs of Ay mice did not differ from lean controls. Sialoadenectomy before and after development of obesity generally reduced the gain of body weights in both sexes of Ay mice but not in ob/ob mice. The content of epidermal growth factor (EGF) in the SMGs was higher in Ay mice and lower in ob/ob mice than their controls. The possible role of EGF in the SMGs in the development of obesity is discussed.

Animals↗

In vivo secretory responses of submandibular glands in streptozotocin-diabetic rats to sympathetic and parasympathetic nerve stimulation.

Submandibular gland responses to sympathetic and parasympathetic nerve stimulation were studied in streptozotocin-diabetic rats. Morphologically, the acinar cells in control glands were relatively uniform in size and contained electron-lucent granules. The granular ducts were distinguished by the presence of electron-dense granules. With the exception of intracellular lipid droplets and the presence of a few autophagosomes in diabetic glands, no consistent differences in acinar cell structure were observed. In contrast, the diameter of the granular ducts and the granule content of their cells were less in diabetic glands. At 3 weeks sympathetic flow rate, salivary protein concentration, and total protein output were unaffected by diabetes. Sympathetic flow rate was greater at 3 months, and the concentration of protein in the saliva was lower. In 6-month diabetic rats flow rate remained increased, but protein concentration and total protein output were reduced. The decrease in salivary protein concentration at 3 and 6 months was accompanied by a reduction in secretory granule release from acinar and granular duct cells. No consistent differences in flow rate, protein concentration, protein output, or secretory granule release were observed following parasympathetic stimulation. We conclude that the effects of diabetes on nerve-stimulated flow rate and protein release depend on the duration of diabetes and the type of stimulation, and are independent of one another.

Animals↗

Characterization and partial purification of rat submandibular gland protein kinase C.

Membrane-bound protein kinase C of rat submandibular gland was characterized and the cytosolic kinase C of the tissue was partially purified. The membrane-bound kinase could be activated by Triton X-100 but not EGTA in the presence of both Ca2+ and phosphatidylserine (PS). The Km values for Ca2+ and PS were 150 microM and 5 micrograms, respectively. Addition of 10(-6) M diacylglycerol resulted in an increased affinity of the kinase for Ca2+ (Km = 10 microM). Phorbol 12,13-dibutyrate activated the kinase in the absence of exogenous Ca2+ and PS, suggesting that adequate amounts of each activator are present in the membrane itself. Polymyxin B inhibited the stimulated kinase C activity in a concentration-dependent manner. This inhibition could be overcome by addition of PS. The cytosolic kinase was partially purified 133-fold by chromatography on columns of DEAE-Sephacel and S-300 Sephacryl. The total kinase activity increased with respect to the kinase activity measured in the starting material with column chromatography, suggesting that an inhibitor is present in the cytosolic fraction of the tissue.

Animals↗

Ultrastructural responses by Golgi apparatus of rat submandibular gland to beta-adrenergic, alpha-adrenergic, and cholinergic stimulation.

Rat submandibular gland tissue pieces were stimulated in vitro for 30 min with a beta-adrenergic agent or a cyclic AMP analog to stimulate protein secretion, or with alpha-adrenergic or cholinergic agents or a Ca2+ ionophore to stimulate fluid secretion. Acinar cells were examined by transmission electron microscopy. In control tissue, acinar cells showed little evidence of secretory activity. The Golgi apparatus was sparse and was associated with a few small, immature secretory granules with fine fibrillar contents. Following secretory granule discharge stimulated by isoproterenol or dibutyryl cyclic AMP, acinar cells were constricted, and had extensive basolateral membrane folding and tightly packed rough endoplasmic reticulum. Golgi complexes were prominent and had multiple small granules with filamentous contents. After stimulation of fluid secretion by alpha-adrenergic agents (epinephrine, phenylephrine), or cholinergic agents (acetylcholine, carbachol, pilocarpine), or a Ca2+ ionophore (A23187), the Golgi apparatus had compact concave cisternae enclosing aggregates of tubulovesicles. Acinar cells were distended, basolateral membranes were expanded, and rough endoplasmic reticulum was dilated and vesiculated.

Acetylcholine↗

Immunohistochemical analysis of DNA synthesis during chronic stimulation with isoproterenol in mouse submandibular gland.

We investigated the numbers of DNA-synthesizing cells in mouse submandibular glands (SMGs) during chronic isoproterenol (IPR) administration, using an immunohistochemical method with anti-bromodeoxyuridine (BrdU) antibodies. Adult and immature female mice were injected with IPR daily or every other day up to a total of seven injections. Whereas hypertrophic enlargement of acinar cells continued with repeated IPR injections, induction of DNA synthesis was transient. With one IPR injection, more than 50% of the acinar cells were induced to synthesize DNA, but after five daily injections the numbers of cells in the DNA synthetic phase decreased and returned to control values. This pattern was basically the same for immature mice and for adult mice stimulated by IPR every other day. The responsiveness of the SMGs of immature mice to IPR was somewhat lower than that of adults. Prolongation of the interval between injections of IPR, compared to daily injections, did not cause sustained induction of DNA synthesis. These data suggest that the hypertrophic enlargement and the induction of DNA synthesis by IPR are mediated through different intracellular pathways after the stimulation of beta-adrenergic receptors, and that the regulatory mechanism for the induction of DNA synthesis is complex.

Age Factors↗

Influence of X-ray irradiation on the ultrastructure of rat submandibular gland striated-duct cells.

Previous investigations have indicated that striated-duct cells react to stimulation with an apocrine secretion, morphologically demonstrated by bleb-like projections of the apical cytoplasm. Since bleb formation as an ultrastructural feature also has been debated and sometimes interpreted as a fixation artifact, it was considered essential to extend the studies by exposing the submandibular gland to X rays to establish whether such treatment would have any influence on the formation of blebs. The material used in the present study consisted of rat submandibular glands exposed to X rays in the range of 200-1800 rad. The glands were examined by both SEM and TEM. The duct cells exposed to 200 rad appeared normal, with no sign of alteration in their ability to produce blebs, whereas duct cells exposed to 750 rad showed no sign of bleb formation. Some of the duct cells exposed to 1800 rad showed considerable morphological changes, consistent with oncotic transformation. The results support the conclusion that bleb formation is a normal morphological feature and not an artifact. This study also indicates that the functional activity of the cells is reduced after exposure to X rays.

Animals↗

Identification of functional receptors for vasoactive intestinal peptide and neurotensin in the human submandibular gland duct cell line, HSG-PA.

The HSG-PA human submandibular gland adenocarcinoma cell line has attracted attention recently as a potentially useful cell culture model for studies of salivary duct cell function and regulation. These cells possess a variety of morphological and biochemical markers found in salivary duct cells. Recently, muscarinic cholinergic receptors coupled to inositol intracellular Ca2+ mobilization (He et al., Eur. J. Physiol., 413 (1989) 505-510) and K+ fluxes (Ship et al., Am. J. Physiol., 259 (1990) C340-C348) have been identified in HSG-PA cells. In this study, we report the presence in these cells of functional receptors for two neuropeptides, vasoactive intestinal peptide (VIP) and neurotensin. Receptors for both peptides were labeled in intact cell radioligand binding studies and exhibited pharmacological profiles similar to receptors found in other tissues. There was close agreement between binding Ki values and the ED50 values for stimulation of second messenger production and modulation of K+ efflux, with all values between 1 and 5 nM. Whereas neurotensin stimulated K+ efflux dramatically, VIP alone had no effect but enhanced the response to neurotensin. These studies thus represent the initial documentation of functional receptors for VIP and neurotensin in a cell line of salivary duct cell origin.

Adenocarcinoma↗

Purification and characterization of kallikrein-like serine proteases from rat submandibular glands.

The purification and characterization of kallikrein-like proteases from rat submandibular glands is described. The proteolytic activity of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME). The purification scheme involved ammonium sulfate precipitation, chromatography on columns of DEAE-Sepharose and Sephadex G-100 and chromatofocusing. Three TAME-hydrolytic activity peaks were eluted from DEAE-Sepharose as unbound fraction (Pool 1), at 125 mM NaCl (Pool 2) and at 250 mM NaCl concentration (Pool 4). Pool 1 further resolved into two protease fractions (1A1 and 1A2), pool 2 into three protease fractions (2A1, 2A2 and 2A3) and pool 4 gave a single major protease peak (4A1) by chromatofocusing on PBE-94. Protease pools 2A2, 2A3, and 4A1 each gave a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular weight of 34 kDa, 46 kDa and 46 kDa respectively. Pools 1A1, 1A2, 2A1 and 2a2 gave a single precipitin line with anti-rat glandular kallikrein antibodies. 2A3 and 4A1 did not react with these antibodies. Synthetic substrates DL-val-leu-arg-pNA and Bz-pro-phe-arg-pNA, specific for kallikrein-like proteases, were hydrolyzed preferentially by 2A3 and 4A1 but were poor substrates for 1A1, 1A2, 2A1 and 2A2.

Amino Acids↗