Alleles from chromosomes 1 and 3 of NOD mice combine to influence Sjögren's syndrome-like autoimmune exocrinopathy.

OBJECTIVE: NOD mice exhibit at least 2 overlapping autoimmune diseases: autoimmune endocrinopathy (Type I, insulin dependent diabetes) and autoimmune exocrinopathy (Sjogren's syndrome, SS). To date, 18 chromosomal regions have been identified that contribute to development of diabetes in NOD mice; however, genetic mapping of similar susceptibility loci for autoimmune exocrinopathy is just beginning. We investigated if these 2 autoimmune diseases share a genetic predisposition. METHODS: Congenic partner strains of NOD and C57BL/6 mice containing defined genetic intervals influencing susceptibility to diabetes (Idd) were screened for histological and biochemical markers for their effect on the development of SS-like disease. Saliva flow rates, protein concentration, amylase activity, and cysteine protease activity were evaluated. RESULTS: In contrast to the nonsusceptible parental C57BL/6 strain, C57BL/6.NOD Idd5 congenic partner strain, containing a genetic region derived from chromosome 1 of the NOD mouse, exhibited pathophysiological characteristics of autoimmune exocrinopathy. Replacement of individual diabetes susceptibility intervals Idd3, Idd5, Idd13, Idd1, and Idd9, as well as a combination of the Idd3, Idd10, and Idd17 intervals, with resistance alleles had little effect on development of autoimmune exocrinopathy. Conversely, NOD mice, in which the chromosome regions containing both Idd5 and Idd3 have been replaced by intervals derived from C57BL mice, exhibit a reduced pathophysiology associated with SS-like autoimmune exocrinopathy. CONCLUSION: Alleles on chromosomes 1 (Idd5) and 3 (Idd3) in combination appear to greatly influence susceptibility and resistance to development of autoimmune exocrinopathy. The association with certain Idd, but not other Idd loci, indicate that genetic overlap is present but probably not inclusive.

Single-molecule imaging of human insulin receptor ectodomain and its Fab complexes.

The insulin receptor (IR) is a four-chain, transmembrane dimer held together by disulfide bonds. To gain information about the molecular envelope and the organization of its domains, single-molecule images of the IR ectodomain and its complexes with three Fabs have been analyzed by electron microscopy. The data indicate that the IR ectodomain resembles a U-shaped prism of approximate dimensions 90 x 80 x 120 A. The width of the cleft (assumed membrane-distal) between the two side arms is sufficient to accommodate ligand. Fab 83-7, which recognizes the cys-rich region of IR, bound halfway up one end of each side arm in a diametrically opposite manner, indicating a twofold axis of symmetry normal to the membrane surface. Fabs 83-14 and 18-44, which have been mapped respectively to the first fibronectin type III domain (residues 469-592) and residues 765-770 in the insert domain, bound near the base of the prism at opposite corners. These images, together with the data from the recently determined 3D structure of the first three domains of the insulin-like growth factor type I receptor, suggest that the IR dimer is organized into two layers with the L1/cys-rich/L2 domains occupying the upper (membrane distal) region of the U-shaped prism and the fibronectin type III domains and the insert domains located predominantly in the membrane-proximal region.

Supersensitivity to serotonin- and histamine-induced arterial contraction following ovariectomy.

The modulating role of estrogens and ovariectomy on coronary artery and thoracic aortic rings was examined in female rabbits. Three treatment groups were studied: (1) control, (2) ovariectomy, and (3) ovariectomy + 17beta-estradiol acetate (40 microg/kg per day, i.m. for 7 days). Coronary artery reactivity was studied in the isolated retrogradely perfused heart. Aortic reactivity was studied using endothelium intact and denuded aortic rings. Concentration-response curves were performed to serotonin (5-HT) and histamine. A 21-fold, a 4.7-fold, and a 5.2-fold increase in sensitivity to 5-HT-induced contraction were observed in the ovariectomy group compared to the control group for coronary artery, intact aortic, and denuded aortic preparations, respectively (P < 0.05 for each comparison). Similarly, 34-fold, 4.9-fold, and 5.0-fold increases in sensitivity to histamine-induced contraction were observed in the ovariectomy group compared to control group for coronary artery, intact aortic, and denuded aortic preparations, respectively (P < 0.05 for each comparison). 17beta-Estradiol administration reversed the supersensitivity to serotonin- and histamine-induced vascular contraction observed following ovariectomy. No differences in EC50 or maximal contraction were noted between control and ovariectomy + estrogen groups. Baseline nitric oxide release and maximal 5-HT- and histamine-induced nitric oxide release from the perfused heart were decreased (P < 0.05) in ovariectomy rabbits compared to control and ovariectomy + estrogen treatment groups. The data demonstrate that (1) reduced autacoid-induced nitrous oxide release following ovariectomy and (2) direct effects upon the vascular smooth muscle contractility, which are probably mediated by altered receptor sensitivity by ovariectomy and estrogen replacement therapy. The information obtained from this study provides additional information regarding possible beneficial actions of estrogen replacement therapy in post-menopausal women.

Excessive synthesis of matrix metalloproteinases in exocrine tissues of NOD mouse models for Sjögren's syndrome.

OBJECTIVE: Matrix metalloproteinases (MMP) and their substrates, components of the extracellular matrix, regulate environmental signals for cellular differentiation and tissue function. Changes in the levels of these enzymes may influence cell survival as well as pathology involving ectopic apoptosis. Using the non-obese diabetic (NOD) mouse model for Sjögren's syndrome, we evaluated the synthesis and expression of MMP in the exocrine target tissues of autoimmunity. METHODS: NOD, immunodeficient NOD-scid, and nondiabetic NOD.B10.H2b mice were evaluated for MMP activity in their saliva and exocrine gland lysates by gelatin zymography and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, changes in protein content of saliva and gland lysates were determined by specific Western blot and by enzymatic activity of amylase and cysteine proteases. Mice continuously treated with the MMP inhibitor GM6001 were evaluated from 7 to 20 weeks of age for the contribution of MMP activity to development of these hallmark biochemical markers of Sjogren's syndrome-like disease of NOD mice. RESULTS: Gelatin zymography of whole saliva and gland lysates indicated the presence of increased proteolytic activity, corresponding to proteins with a molecular mass ranging from 50 to 95 kDa, in the saliva of older (> 20 weeks of age) NOD mice as well as NOD.B10.H2b and NOD-scid mice compared to BALB/c controls. Elevated steady state levels of mRNA transcripts for the gelatinases MMP-2 and MMP-9 were detected in total RNA extracted from parotid and submandibular glands by RT-PCR. Despite prophylactic injection of the broad spectrum MMP inhibitor GM6001 into mice beginning at 7 weeks of age and continuing to 20 weeks, development of the autoimmune exocrinopathy was neither stopped nor retarded. CONCLUSION: These observations suggest that excessive MMP activity is associated with autoimmune Sjögren's syndrome-like disease in NOD mice. However, a possible contribution by increased MMP activity in initiation and progression of this autoimmune disease is yet to be elucidated.

Inappropriate apoptosis of salivary and lacrimal gland epithelium of immunodeficient NOD-scid mice.

OBJECTIVE: The lesion is Sjögren's syndrome consists of lymphocytic infiltration and has a pathology characteristic of the potential apoptotic death of salivary gland secretory epithelial cells. To examine the role of the glandular epithelial cells in the pathogenesis of autoimmune exocrinopathy, we studied Fas and Fas ligand (FasL) expression and quantitated the levels of apoptosis in salivary and lacrimal glands from NOD and NOD-scid mice, an animal model that develops a Sjögren's syndrome-like pathology. METHODS: The parotid, submandibular and lacrimal tissues of NOD, NOD-scid, and BALB/c mice were evaluated by immunohistochemical analysis for the expression of Fas and FasL. Nuclear fragmentation of DNA from the epithelial cells of exocrine tissues was evaluated by the terminal UTP nucleotide end labeling method (TUNEL). Messenger RNA was isolated from 8 and 18 week old mice and was analyzed by the reverse transcription-polymerase chain reaction (RT-PCR) for the expression of Fas and FasL. RESULTS: We found suggestive evidence that apoptosis of the secretory epithelial cells occurs in both NOD and NOD-scid mice despite the lack of T- and B-lymphocytes in the latter. FasL mRNA and cell surface protein were expressed in salivary and lacrimal gland epithelial cells from 8 and 18 week old NOD, NOD-scid, and BALB/c mice. Fas protein and mRNA were expressed only in the exocrine glands from 18 week old NOD and NOD-scid mice. Glandular secretory epithelial cell apoptosis was elevated in both NOD and NOD-scid mice, however; there was little evidence of apoptosis in the control strain of BALB/c mice. CONCLUSION: These results suggest a potential apoptotic process dependent on Fas:FasL interactions occurring in NOD-scid glandular secretory epithelial cells in the absence of lymphocytic infiltration.

Salivary gland changes in the NOD mouse model for Sjögren's syndrome: is there a non-immune genetic trigger?

Sjögren's syndrome is a systemic autoimmune disease characterized by patient complaints of oral and ocular dryness accompanied by clinical observations of a progressive loss of salivary and lacrimal function, related to the presence of a focal, periductal leukocyte infiltrate. Progress in understanding the mechanisms involved in the development of autoimmune diseases in general, and Sjögren's syndrome specifically, has been generated as a result of renewed interest in animal models, such as the NOD mouse, which mimics autoimmune sialoadenitis. Biochemical analyses have indicated that the salivary glands have reduced beta-adrenergic, muscarinic, and neuropeptide signal transduction responses that correlate to reduced receptor density and the appearance of autoantibodies directed against these and other cell surface proteins. Using the NOD-scid mouse (lacking functional B- and T-lymphocytes) it has been determined that salivary flow rates are normal; however, these animals show abnormal changes in protein biosynthesis with increasing age. Histological evaluation of the submandibular gland from older NOD-scid mice revealed a loss of acinar cells accompanied by a potential increase in the ductal cell component of the tissue. Consistent with this finding, we recently have observed increased levels of cell death-associated cysteine proteases in the submandibular glands of 20 week NOD and NOD-scid mice but not in BALB/c and young NOD controls. Other novel protease activity was detected in the parotid and submandibular glands from NOD mice, which were able to generate the aberrantly processed PSP from purified BALB/c protein. Taken together, these data paint a complex picture of the development of Sjögren's syndrome-like disease in the NOD mouse model. The presence of activated lymphocytes appears to be necessary for the ultimate loss of exocrine gland function, potentially through the loss of tolerance to glandular proteins. However, the findings of high levels of apoptosis and aberrant protein expression in the submandibular gland in the absence of an immune response (NOD-scid) suggests that genetic alterations in glandular homeostasis involving the death program contribute to disease progression or even the initial trigger of autoimmunity.

Transfer of human serum IgG to nonobese diabetic Igmu null mice reveals a role for autoantibodies in the loss of secretory function of exocrine tissues in Sjögren's syndrome.

The NOD (nonobese diabetic) mouse has been studied as an animal model for autoimmune insulin-dependent diabetes and Sjögren's syndrome. NOD.Igmu null mice, which lack functional B lymphocytes, develop progressive histopathologic lesions of the submandibular and lachrymal glands similar to NOD mice, but in the absence of autoimmune insulitis and diabetes. Despite the focal appearance of T cells in salivary and lachrymal tissues, NOD.Igmu null mice fail to lose secretory function as determined by stimulation of the muscarinic/cholinergic receptor by the agonist pilocarpine, suggesting a role for B cell autoantibodies in mediating exocrine dryness. Infusion of purified serum IgG or F(ab')2 fragments from parental NOD mice or human primary Sjögren's syndrome patients, but not serum IgG from healthy controls, alters stimulated saliva production, an observation consistent with antibody binding to neural receptors. Furthermore, human patient IgG fractions competitively inhibited the binding of the muscarinic receptor agonist, [3H]quinuclidinyl benzilate, to salivary gland membranes. This autoantibody activity is lost after preadsorption with intact salivary cells. These findings indicate that autoantibodies play an important part in the functional impairment of secretory processes seen in connection with the autoimmune exocrinopathy of Sjögren's syndrome.

Characterization of the changing lymphocyte populations and cytokine expression in the exocrine tissues of autoimmune NOD mice.

NOD mice develop chronic lymphocytic invasion of the pancreas, submandibular, and lacrimal glands leading to loss of insulin secretion, salivary flow, and tear production. In this study, we have used flow cytometric analyses and RT-PCR to track glandular lymphocyte populations and cytokine expression spanning the initiation of autoimmune infiltration through the development of widespread autoimmune destruction of the salivary and lacrimal glands of NOD mice. Results demonstrate a predominance of CD4+ to CD8+ lymphocytes and a similar predominance of T-cells versus B-cells in both the submandibular and lacrimal gland infiltrates. A temporal increase in memory (CD3+CD45RBlo) T-cells was also detected; however, naive (CD3+CD45RBhi) T-cell populations as well as a CD3+, CD4-/CD8- double negative population were also present. In addition, a skewing of the TCR Vbeta repertoire toward Vbeta6+ and Vbeta8+ lymphocytes was evident in both glandular infiltrates. Analyses of cytokine mRNA expression in the submandibular glands demonstrated an increase between 12 and 16 wk of age of several proinflammatory cytokines including IL-1beta, IL-6, IL-7, IL-10, IFNgamma, TNFalpha, and inducible Nitric Oxide Synthase (iNOS). IL-4 synthesis was notably absent in both tissues. Cytokine mRNA transcripts detected in lacrimal tissue were similar to those seen in the submandibular glands but appeared both earlier and more intensely. These findings depict the progressive development of autoimmune exocrinopathy and can be used as a foundation to explore the similarities and potential differences in the immunopathogenic lesions of several distinct tissues within the same host.

Ovariectomy and estrogen-induced alterations in myocardial contractility in female rabbits: role of the L-type calcium channel.

The effects of ovariectomy and estrogen replacement on myocardial contractility were examined in female rabbits. Ovariectomy failed to alter left ventricular mass, papillary muscle cross-sectional area or isometric force. Estrogen replacement after ovariectomy (0.15 microg/kg/day i.m. 17beta-estradiol acetate for 7 days) increased left ventricular mass and papillary muscle mass, and reduced isometric force compared to control and ovariectomy groups. Ovariectomy did not alter increased isometric force with isoproterenol, but decreased the ED50 for Bay K8644 (compared to control and estrogen groups). Estrogen replacement increased the ED50 for isoproterenol- and Bay K8644-induced isometric force compared to control and ovariectomy groups. Ovariectomy increased and estrogen replacement decreased isometric force associated with increased Ca++o. Acute exposure to 17beta-estradiol or diethylstilbesterol (10(-7) M, 10(-6) M) failed to alter isometric force in control papillary muscles. Estrogen replacement reduced the number, but not the dissociation constant for 3H-nitrendipine binding in plasma membrane preparations (compared to ovariectomy and control groups). Peak L-type calcium currents in isolated ventricular myocytes from the three treatment groups were not significantly different. The data are consistent with an ovariectomy-induced increase and estrogen-induced decrease in L-type calcium channel density in rabbit myocardium. Estrogen-induced alterations in L-type calcium channel expression and contractility are subsequently modified by estrogen-induced cardiac hypertrophy.

A novel NOD-derived murine model of primary Sjögren's syndrome.

OBJECTIVE: The appearance of autoimmune diabetes prior to autoimmune exocrinopathy in the NOD mouse suggests that it is an excellent model of secondary, but not primary, autoimmune sicca complications. Since the unique major histocompatibility complex (MHC) I-A(g7) expression in NOD mice is essential for the development of insulitis and diabetes in these animals, we investigated exocrine gland function in NOD.B10.H2b mice, which have an MHC congenic to NOD, as a potential model for primary Sjögren's syndrome (SS). METHODS: Histopathologic manifestations of lymphocytic infiltrates into the pancreas and exocrine tissues were examined by light microscopy. Sera were evaluated for the presence of antinuclear antibodies. Saliva, tears, and gland lysates were evaluated for total volume and protein concentration, the aberrant expression and processing of parotid secretory protein, and cysteine protease activity. RESULTS: NOD.B10.H2b mice exhibited the exocrine gland lymphocytic infiltration typical of the SS-like disease and dysfunction observed in NOD mice, but without the insulitis and diabetes. These mice additionally expressed elevated levels of cysteine protease activity (a measure of apoptotic activity) and abnormal expression and cleavage of parotid secretory protein in the submandibular tissues. CONCLUSION: The results of this study suggest that the unique NOD MHC I-A(g7) is not essential for exocrine tissue autoimmunity. Furthermore, the findings indicate that sicca syndrome occurs independently of autoimmune diabetes and that the congenic NOD.B10.H2b mouse represents a novel murine model of primary SS.

The disulfide bonds in the C-terminal domains of the human insulin receptor ectodomain.

The human insulin receptor is a homodimer consisting of two monomers linked by disulfide bonds. Each monomer comprises an alpha-chain that is entirely extracellular and a beta-chain that spans the cell membrane. The alpha-chain has a total of 37 cysteine residues, most of which form intrachain disulfide bonds, whereas the beta-chain contains 10 cysteine residues, four of which are in the extracellular region. There are two classes of disulfide bonds in the insulin receptor, those that can be reduced under mild reducing conditions to give alpha-beta monomers (class I) and those that require stronger reducing conditions (class II). The number of class I disulfides is small and includes the alpha-alpha dimer bond Cys524. In this report we describe the use of cyanogen bromide and protease digestion of the exon 11 plus form of the receptor ectodomain to identify disulfide linkages between the beta-chain residues Cys798 and Cys807 and between the alpha-chain Cys647 and the beta-chain Cys872. The latter bond is the sole alpha-beta link in the molecule and implies a side-by-side alignment of the two fibronectin III domains of the receptor. Also presented is evidence for additional alpha-alpha dimer bond(s) involving at least one of the cysteine residues of the triplet at positions 682, 683, and 685. Evidence is also presented to show that Cys884 exists as a buried thiol in the soluble ectodomain.

Assessment of bromhexine as a treatment regimen in Sjögren's syndrome-like disease in the NOD (non-obese diabetic) mouse.

OBJECTIVE: Bromhexine has been reported to alleviate the xerostomia and xerophthalmia associated with secondary Sjögren's syndrome. The aim of this study was to determine if it might prove useful in the treatment of Sjögren's syndrome-like disease of the NOD mouse model for autoimmune sialoadenitis. METHODS: Groups of mice were divided into sets receiving 60 mg/kg bromhexine in drinking water and control pair-fed animals. The efficacy of drug treatment was assessed by weekly measurement of stimulated saliva volumes, protein concentration, and amylase activity. At termination (20 weeks) submandibular and lacrimal glands were removed to assess the levels of lymphocytic infiltration by histological evaluation under light microscopy. RESULTS: Control and bromhexine-treated groups of mice showed no difference in the loss or rate of reduction in stimulated saliva flow over the 12 weeks of treatment. No differences were noted in the protein concentration and amylase loss with increasing age of the animals. Similar temporal changes in total protein profiles and aberrant expression of the 20 kDa parotid secretory protein isoform were observed by SDS-polyacrylamide gel profiles and Western bolt analysis. Histological evaluation of exocrine gland sections failed to detect any reduction in focal lymphocyte infiltration. CONCLUSION: Bromhexine therapy did not alter the development or severity of Sjögren's syndrome-like disease in the NOD mouse model for autoimmune sialoadenitis.

Effect of 17-beta estradiol in the rabbit: endothelium-dependent and -independent mechanisms of vascular relaxation.

Short-term estrogen administration has been independently proposed to produce arterial vasodilation by both an indirect mechanism and a direct mechanism (inhibition of calcium entry though the L-type calcium channel). The proposed contributions of such diverse mechanisms to the vascular actions of 17beta-estradiol were examined in perfused hearts and in aortic ring sections isolated from female rabbits. In isolated rabbit hearts retrogradely perfused with Tyrode's solution, concentration-response curves to 17beta-estradiol (10(-9)-10(-5) M) were performed under control conditions and during perfusion with Bay K8644 (10(-7) M). 17beta-Estradiol produced a concentration-dependent decrease in coronary vascular resistance proportional to nitric oxide (NO) release in the presence and absence of Bay K8644. The addition of N(G)-nitro-L-arginine methyl ester (L-NAME; 10(-4) M) to the perfusate (a) completely inhibited NO formation, (b) produced a 2.3-fold and 1.55-fold rightward shift in the concentration-response curve to 17beta-estradiol for Bay K8644 treated and control hearts, respectively, and (c) failed to prevent coronary artery vasodilation. In isolated aortic rings contracted with Bay K8644, 17beta-estradiol (10(-5) M) relaxed both intact (58%) and denuded (54%) aortic rings. L-NAME (10(-4) M) completely blocked NO release in intact rings but did not prevent relaxation in denuded aortic rings. The data demonstrate (a) an endothelium-dependent relaxation by 17beta-estradiol, coincident with NO formation and suppressed by L-NAME, and (b) a direct relaxation of aortic and coronary smooth muscle independent of NO formation at higher 17beta-estradiol concentrations.

Elevated levels of cysteine protease activity in saliva and salivary glands of the nonobese diabetic (NOD) mouse model for Sjögren syndrome.

Nonobese diabetic (NOD) mice develop an anti-exocrine gland pathology similar to human Sjögren syndrome. Recently, we demonstrated that NOD-scid mice develop severe loss of submandibular acinar cells with concomitant appearance of abnormal isoforms of salivary proteins suggesting de novo enzymatic cleavage. Because these changes may indicate activation of apoptotic proteases, we examined saliva and salivary tissue for cysteine protease activity. Cysteine protease activities were elevated in saliva and gland lysates from 20-week-old NOD and NOD-scid mice as compared with age- and sex-matched BALB/c or 8-week-old NOD mice. This activity appeared in the submandibular glands, but not in the parotid glands. Western blot analyses using antibodies directed against specific apoptotic proteases (interleukin 1beta converting enzyme, Nedd-2, and Apopain/CPP 32) confirmed these findings. Submandibular glands from NOD-scid mice exhibited the greatest increase in proteolytic activity, indicating that infiltrating leukocytes are not responsible for these changes. Western blot analyses also failed to reveal changes in the levels of cystatins (saliva proteins that inhibit protease activity). Thus, increased cysteine protease activity appears to be directly related to submandibular acinar cell loss in NOD-scid mice involving the apoptotic pathway. Additional protease activity in saliva and gland lysates of older NOD and NOD-scid mice, apparently mutually distinct from cysteine proteases, generated an enzymatically cleaved parotid secretory protein. We suggest, therefore, that proteolytic enzyme activity contributes to loss of exocrine gland tolerance by generating abnormally processed protein constituents.

PSP expression in murine lacrimal glands and function as a bacteria binding protein in exocrine secretions.

Nonobese diabetic (NOD) mice, an animal model for type I autoimmune diabetes and autoimmune sialoadenitis, abnormally express parotid secretory protein (PSP) in the submandibular glands (Robinson, C. P., H. Yamamoto, A. B. Peck, and M. G. Humphreys-Beher. Clin. Immunol. Immunopathol. 79: 50-59, 1996). To evaluate possible PSP gene dysregulation in the NOD mouse, we have examined a number of organs and tissues for PSP mRNA transcripts and protein expression. Results indicate that PSP is produced in the lacrimal glands of NOD mice as well as most laboratory mouse strains. Although purified salivary PSP from C3H/HeJ or BALB/c mice fails to affect amylase enzyme activity in in vitro assays, PSP bound to whole bacteria in a Zn2+-dependent manner. Additionally, radiolabeled protein bound to specific bacterial membrane proteins using a ligand binding assay. PSP gene transcription, but not protein production, was observed in the heart and pancreas from NOD mice, indicating abnormal transcription of the PSP gene. Sequence analysis of PSP cDNA from NOD mice revealed numerous base differences (compared with the published PSP sequence) capable of leading to significant amino acid substitutions, suggestive of strain-specific differences for the protein in mice. Together these results suggest that there exists in the NOD mouse a dysregulation of PSP transcription in various tissues. However, except for C3H/HeJ mice, PSP appears as a normal product of the lacrimal glands where, as in saliva, it may function as a nonimmune antimicrobial agent in the protection of tissue surfaces exposed to the external environment.

Detection of alterations in the levels of neuropeptides and salivary gland responses in the non-obese diabetic mouse model for autoimmune sialoadenitis.

The salivary glands of non-obese diabetic (NOD) mice and BALB/c controls were evaluated for the stimulatory effects of the following neuropeptides; substance P (SP), vasoactive intestinal polypeptide (VIP), and neuropeptide Y (NPY). Injection of either of the three neuropeptides in combination with the muscarinic-cholinergic agonist pilocarpine increased saliva flow rates in BALB/c mice while there was no observable augmentation to flow rates in pre-diabetic or diabetic NOD mice. Small increases in protein content of the stimulated saliva were observed in the BALB/c group of animals with the injection of any of the above neuropeptides in combination with pilocarpine. In pre-diabetic NOD animals, only VIP and NPY increased the protein content-ratio above pilocarpine alone. Radioimmunoassay determination of neuropeptide concentrations in the submandibular and parotid glands revealed reduced levels of SP with diabetes onset as compared with pre-diabetic NOD or BALB/c mice. The levels of NPY were similar between BALB/c and NOD animals except in the pre-diabetic parotid gland where NPY concentrations were 1.3-fold greater. On the other hand, VIP concentrations were substantially reduced in the submandibular gland of NOD mice, while in the parotid gland neuropeptide levels were evaluated 3.8-fold relative to BALB/c controls. Immunohistochemical staining of the parotid and submandibular glands for SP revealed primarily ductal cell staining which was reduced with diabetes onset in NOD animals. These findings further define the sialoadenitis observed in NOD mice to be due, in part, to a general loss of neurotransmitter responsiveness on the part of salivary gland cells.

Ricin depresses cardiac function in the rabbit heart.

Ricin, at toxic glycoprotein from the castor bean, causes myocardial hemorrhage and a decrease in blood pressure. We studied the effects of ricin on myocardial function in the isolated rabbit heart. Rabbits were given 0.22 micrograms/kg of ricin i.v. and 48 hr later, the heart was isolated and retrogradely perfused through the aorta with Tyrode's solution. A latex balloon was inserted into the left ventricle and isovolumic left ventricular function curves were generated. Left ventricular developed pressure (LVDP), heart rate, coronary artery flow, left ventricular end diastolic pressure, myocardial oxygen consumption, oxygen extraction (a - vO2), and contractility (+dp/dt) were measured over a range of left ventricular volumes. Dose-response curves to isoproterenol (10(-9)-10(-8) M) and phenylephrine (10(-9)-10(-6) M) were also obtained. Compared to the control group, ricin pretreatment markedly decreased ventricular compliance (p < 0.01), diminished maximum left ventricular developed pressure (p < 0.05), and reduced maximal +dp/dt (p < 0.05). Myocardial oxygen consumption, heart rate, electrocardiographic PR, QRS, and QT intervals were not different in control and ricin treatment groups. Ricin did not significantly alter the inotropic or chronotropic responses to isoproterenol and phenylephrine. The results from the binding studies showed that ricin neither reduced beta-adrenergic receptor numbers nor altered the dissociation constant. thus, ricin reduced both systolic (LVDP and +dp/dt) and diastolic (compliance) left ventricular functions, perhaps due to increased vascular permeability, without altering responses to the alpha- and beta-adrenoceptor agonists phenylephrine and isoproterenol.

Competitive RNA templates for detection and quantitation of growth factors, cytokines, extracellular matrix components and matrix metalloproteinases by RT-PCR.

Detection of low-abundance mRNAs by reverse transcription-polymerase chain reaction (RT-PCR) has become a standard technique to determine gene expression by tissues and cells in culture. The ability to determine relative or absolute copy number of specific mRNAs has been difficult due to inadequate internal standards to control for sample-to-sample variation. The use of a synthetic RNA standard with identical sequences to the PCR primers allows reproducible quantitation between samples and assays. By designing multi-sequence templates, several specific mRNAs can be quantitated using a single template. Addition of multiple templates to a single RT reaction allows the quantitation of a large number of targets from as little as 4 micrograms of total RNA. In this report, we present a series of seven primer/template systems to detect and quantitate 52 specific messages, including 26 growth factors and receptors, 8 extracellular matrix components, 10 matrix-modifying enzymes and their inhibitors and 8 cytokines.