Normal PSS Research Articles

Immune infiltration analysis reveals immune cell signatures in salivary gland tissue of primary Sjögren's syndrome.

Mouse models are the basis for primary Sjögren's syndrome (pSS) research. However, the depth of comparisons between mice and humans in salivary gland (SG) immune cells remains limited. The gene expression profiles of SGs from normal subjects and pSS patients were downloaded from the Gene Expression Comprehensive Database. The proportion of infiltrating immune cell subsets was then assessed by cell type identification by estimating relative subsets of RNA transcripts (CIBERSORT). An experimental Sjögren's syndrome (ESS) mouse model was successfully constructed using SG protein. Based on mouse SG tissue RNA-Seq data, the seq-ImmuCC model was used to quantitatively analyze the compositional ratios of 10 immune cells in pSS patients and mouse model SG tissues. Computed and obtained 31 human data samples using the CIBERSORT deconvolution method. The immune cell infiltration results showed that, compared to normal human SG tissue, the content of gamma delta T cells was significantly different from naive CD4+ T cells and significantly increased, while the plasma cell content decreased. Principal component analysis indicated differences in immune cell infiltration between pSS patients and normal subjects. Meanwhile, for ESS model mouse data analysis, we found that the proportion of macrophages increased, while the proportion of CD4+ T cells, B cells, and monocytes decreased. Furthermore, we found that the proportion of monocytes was decreased, while the proportion of macrophages was increased in the SG tissues of pSS patients and model mice. The infiltration of CD4+ T, CD8+ T, and B cells also showed some differences. We comprehensively analyzed SG immune infiltration in pSS patients and model mice. We demonstrated conserved and nonconserved aspects of the immune system in mice and humans at the level of immune cells to help explain the primary regulation of immune mechanisms during the development of Sjögren's syndrome.

English

The effect of extracellular calcium on isolated uterine muscle contractions stimulated by acetone leaf extract of Anogeissus leiocarpa was investigated in a rat. About 12 m segment of the uterine muscle strip was mounted initially in a thermostatically regulated organ bath (37°C) containing normal Physiological Salt Solution (PSS; De Jalon) and later inphysiological salt solution devoid of CaCl2. The extract contracted the uterine muscle preparation in a concentration-related manner in normal PSS, with 0.53 mg/ml as the lowest active concentration. This contractile response was abolished by the calcium channel blocker, verapamil HCl (2 µg/ml). The extract however, did not evoke any contractile activity on the isolated tissue in a calcium-free media (PSS), even when caffeine (2 mmol) known to release calcium via the calcium induced-calcium release (CICR) mechanism was added to the perfusate. The results demonstrated the requirement for extracellular calcium for the extract mediated contractions and the inability of the extract to access calcium from intracellular storage sites such as the sarcoplasmic reticulum and the mitochondria. Therefore, the influx of Ca2+ into the cell cytosol is a pre-requisite for the extract-mediated uterine muscle contraction. Key word: Anogeissus leiocarpa, thermostatically organ bath, verapamil, caffeine, uterus.

Effect of the aqueous extract of Chrysanthellum indicum on calcium mobilization and activation of rat portal vein

The effects of the aqueous extract of Chrysanthellum indicum (CI) on calcium activation and mobilization were studied using the rat portal vein. The extract caused a concentration-dependent contraction of the portal vein. KCl (80 mM), norepinephrine (NA, 10 −6 M) and CI (4 mg/ml) evoked sustained contraction of the portal vein. In Ca-free medium (with EGTA) the contractions evoked by these agents were reduced significantly. The times-to-peak of KCl, NA and CI were similar -in normal PSS, but in Ca-free medium the times-to-peak for KCl and CI were greatly increased. The contractions induced by CI were not inhibited by chlorpropamide and prazosin, but were blocked by verapamil. The data obtained suggest that the aqueous extract of CI utilizes extracellular calcium pools to bring about contractile response and this effect might be mediated through the activation of potential-sensitive channels.

Effects of pH on contraction and Ca2+ mobilization in vascular smooth muscles of the rabbit basilar artery.

The present study was undertaken to investigate the effects of extracellular pH (pHe) and intracellular pH (pHi) on 5-hydroxytryptamine (5-HT)-induced contraction and Ca2+ mobilization in vascular smooth muscles. Strip preparations of the rabbit basilar artery without endothelium were loaded with 40 microM fura-2-AM and 2 microM BCECF-AM and mounted in an organ bath. The isometric tension was recorded by using a force displacement transducer. Administration of 5-HT caused dose-dependent contraction in the rabbit basilar arteries. Acidification of pHe from 7.40 to 6.90 reduced the 5-HT-induced contraction and [Ca2+]i transients. Alkalinization of pHe from 7.40 to 7.90, on the other hand, enhanced the contraction and elevation of [Ca2+]i. In the other series of experiments, pHi (7.12 in normal PSS) was selectively altered by adding either butyric acid or trimethylamine. Intracellular acidification (pHi = 6.89) and alkalinization (pHi = 7.35) without changes in pHe produced qualitatively similar effects to those caused by extracellular acidification and alkalinization, respectively. Ca-sensitivity, which is defined as Deltatension/Delta[Ca2+]i, was not affected by the alteration of pHe nor pHi. In the Ca2+-free solution, the addition of 5-HT produced transient increases in [Ca2+]i and isometric tension that were much smaller than those in the normal physiological salt solution. The 5-HT-induced responses of [Ca2+]i and tension in the Ca2+-free solution were not affected by acidification nor alkalinization. These results suggest that a 5-HT-induced contraction is significantly modulated by pH through changing the [Ca2+]i transients, and that the change of pHi plays, at least in part, a role in the alteration of 5-HT-induced contraction resulting from acidosis or alkalosis in the rabbit basilar artery.

Regional differences in α1-adrenoceptor subtypes and mechanisms in rabbit arteries

Contractility mediated through α 1-adrenoceptor subtypes and the maximum binding site ( B max value) and the dissociation constant ( K d value) for [ 125 I] ]HEAT ( [ 125 I] iodo-2-( β-(4-hydroxyphenyl)ethylaminomethyl)tetralone) were determined in the following rabbit arteries: thoracic and abdominal aorta, mesenteric, renal and iliac arteries, and the α 1-adrenoceptor subtypes mediating contractile mechanisms in vascular smooth muscle were studied. The p D 2 values for norepinephrine differed considerably among the arteries in the presence of nicardipine (10 −5 M), while the p A 2 values for 5-methylurapidil against norepinephrine were identical at low affinity in all the arteries used. In Ca 2+-free physiological saline solution (Ca 2+-free PSS), the p A 2 values for 5-methylurapidil were also similar except for the renal artery, in which there were no stable contractions. In normal PSS, the concentration–response curves for norepinephrine with chloroethylclonidine-pretreatment were shifted to the right (p D 2 values of 5.58, 5.70, 5.74, 5.98 and 6.38 for thoracic and abdominal aorta, mesenteric, renal and iliac arteries, respectively). In the [ 125 I] HEAT binding study using membrane preparations obtained from chloroethylclonidine-treated strips, the B max values (33.2–105.2 fmol/mg protein) for [ 125 I] HEAT varied considerably among arteries, while the K d values (0.20–0.26 nM) were identical. The logarithm of B max values is proportional to the p D 2 values for norepinephrine (slope=0.69, r=0.961). These observations suggest that the regional differences in potency (p D 2 value) of the α 1-adrenoceptor agonist, norepinephrine, are a result of the differences in population and density of α 1-adrenoceptor subtypes in rabbit arteries.

Expression and function of α1-adrenoceptor subtypes in the porcine renal artery

We investigated the expression and function of α 1-adrenoceptor subtypes in the porcine renal artery. Reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequencing indicated that the mRNAs for α 1a- and α 1b-adrenoceptors were expressed in the porcine renal artery. Chloroethylclonidine, an α 1B- and α 1D-adrenoceptor antagonist, partially inhibited the phenylephrine-induced contraction, while 3 nM BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione dihydrochloride), an α 1D-adrenoceptor antagonist, had no effect. In contrast, 5-methylurapidil, an α 1A-adrenoceptor antagonist, induced a rightward shift of the phenylephrine concentration–response curve. The simultaneous measurement of cytosolic Ca 2+ concentration ([Ca 2+] i) and tension revealed that chloroethylclonidine pretreatment abolished the phenylephrine-induced increases in [Ca 2+] i and tension in the Ca 2+-free solution. The application of 5-methylurapidil (3 nM) to the chloroethylclonidine-pretreated strips completely inhibited the 3 μM phenylephrine-induced [Ca 2+] i and tension increase in normal PSS. We concluded that both α 1A- and α 1B-adrenoceptors mediate the phenylephrine-induced contraction of the porcine renal artery accompanied by an increase in [Ca 2+] i, and that α 1A-adrenoceptors cause Ca 2+ influx whereas α 1B-adrenoceptors mainly mediate Ca 2+ release.

Release of the antioxidants ascorbate and urate from a nitrergically-innervated smooth muscle.

1. The main object of the present study was to determine whether ascorbate, an antioxidant which has been shown to protect nitric oxide (NO) from attack by scavenger molecules, might be released from nitrergically-innervated smooth muscle; ascorbate release from the rat anococcygeus was measured by use of h.p.l.c. with electrochemical detection. 2. Incubation of rat anococcygeus muscles in normal physiological salt solution (PSS; 30 min) resulted in release of ascorbate into the bathing medium (7.7 +/- 0.9 nmol g-1 tissue). This release was increased by 96% when muscles were incubated in high K+ (70 mM) PSS. The resting release of ascorbate was unaffected by tetrodotoxin (TTX; 1 microM), omega-conotoxin GVIA (10 nM) or omission of calcium ions from the PSS (with addition of 0.2 mM EGTA), but all three procedures attenuated the increased release observed under depolarizing conditions. Resting release of ascorbate was unaffected by glutamate (100 microM), aspartate (100 microM), gamma-aminobutyric acid (100 microM) or carbachol (50 microM). 3. A second h.p.l.c. peak, which always preceded the ascorbate peak, was identified as urate. Urate release from the anococcygeus, following 30 min incubation in normal PSS, was 64.6 +/- 12.7 nmol g-1 tissue but, unlike ascorbate, urate release was unchanged in high K+ PSS. In functional experiments, urate (100-400 microM) partially protected NO (15 microM)-induced relaxations of the rat anococcygeus from inhibition by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; 50 microM), but not from inhibition by hydroquinone or duroquinone (both 100 microM). 4. Muscles chemically sympathectomized with 6-hydroxydopamine (6-OHDA, 500 microM; 2 h) still exhibited release of ascorbate (2.5 +/- 0.4 nmol g-1 tissue) and urate (22.2 +/- 2.9 nmol g-1 tissue); in both cases the release was similar to that observed in time-matched control tissues not exposed to 6-OHDA. High K+ PSS produced a TTX-sensitive increase in release of ascorbate, but not urate, from 6-OHDA-treated muscles. 5. The results demonstrate that significant amounts of ascorbate and urate are released from the rat anococcygeus muscle. Ascorbate, but not urate, release appears to be enhanced by activation of nerves which are resistant to 6-OHDA pretreatment. Since both antioxidants can protect NO from attack by scavenger molecules, their release in nitrergically-innervated tissues may be important for the provision of the correct redox environment to allow NO to fulfill its proposed neurotransmitter role.

Mechanism of endothelium‐dependent relaxation induced by substance P in the coronary artery of the pig

1. Using front-surface fluorometry of fura-2-loaded porcine coronary arterial strips with the endothelium intact, we investigated the mechanisms of vasorelaxation induced by substance P (SP). Fura-2 fluorescence signals which indicated the cytosolic Ca2+-concentration ([Ca2+]i), were observed to arise exclusively from teh smooth muscle cells in these strips. 2. During the contractions induced by U46619 (100 nM), a thromboxane A2 analogue, an SP-induced endothelium-dependent, biphasic vasorelaxation was observed, which consisted of an initial rapid relaxation phase followed by a sustained phase, with a transient decrease in [Ca2+]i. Pretreatment with indomethacin (Ind) had no effect on the SP-induced relaxation; however, pretreatment with NG-nitro-L-arginine (L-NOARG) partially, but significantly inhibited the decrease in both the [Ca2+]i and tension abolished. Thus, part of the relaxation was considered to be mediated by L-NOARG-sensitive relaxing factor (endothelium-derived relaxing factor: EDRF). 3. During the 40 mM K+-depolarization-induced contraction which may eliminate the effects of endothelium-derived hyperpolarizing factor (EDRF), the vasorelaxation reduced by SP was completely inhibited by L-NOARG. 4. During the vasorelaxation induced SP, the [Ca2+]i-tension relationships shifted to the right of the contractions induced by either U46619 or high K+-depolarization. 5. Using front-surface fluorometry of fura-2 loaded porcine aortic valvular strips, we examined the effects of SP on [Ca2+]i in endothelial cells in situ. SP induced a rapid increase in [Ca2+]i of endothelial cells in situ followed by a small sustained phase in normal PSS (5.9 mM K+). The increase in extracellular K+ had no apparent effect on the SP-induced [Ca2+]i elevation of endothelial cells.

Role of GTP-protein and endothelium in contraction induced by ethanol in pig coronary artery.

1. We examined the effects of ethanol on the contractility of strips of porcine coronary artery, with and without endothelium, and following permeabilization with alpha-toxin, and of aortic valvular endothelial cells, in situ. Changes in cytosolic Ca2+ concentration ([Ca2+]i) of the coronary artery smooth muscle cells and of the valvular endothelial cells were monitored using front-surface fluorometry of the calcium indicator dye, fura-2. In permeabilized preparations, [Ca2+]i was clamped using 10 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetra ace tic acid (EGTA) and 10 microM A23187 (a calcium ionophore). 2. The strips without endothelium were placed in normal physiological salt solution (normal PSS) in the presence of ethanol (100-1000 mM). There were dose-dependent increases in [Ca2+]i and a rapid sustained rise in tension. In Ca(2+)-free PSS, ethanol increased [Ca2+]i and tension, similar to, but much smaller than, findings with normal PSS. 3. For a given change in [Ca2+]i induced by ethanol, the developed tension was greater than that observed during contractions induced by high [K+]o. Thus, the [Ca2+]-tension curve for ethanol was shifted to the left of that for high [K+]o. The [Ca2+]-tension curve for the contraction induced by ethanol in the absence of extracellular Ca2+ was shifted further to the left from that obtained in the presence of [Ca2+]o. 4. The mechanisms involved in this Ca(2+)-sensitizing effect of ethanol were investigated using alpha-toxin-permeabilized coronary medial strips. Ethanol increased the tension development, in a concentration-dependent manner, at a fixed concentration of Ca2+ (pCa = 6.3) in the presence of guanosine-5'-triphosphate (GTP), an effect antagonized by guanosine-5'-O-(beta-thiodiphosphate) (GDP beta S), a non-hydrolysable GDP analogue. 5. With intact endothelium, the ethanol-induced tension development was markedly reduced, although inhibition in the increase in [Ca2+]i was slight. The [Ca2+]-tension relationship of this contraction overlapped with that obtained with high [K+]o-induced contraction and was shifted to the right from that obtained in the absence of the endothelium. This endothelium-dependent reduction of [Ca2+]i and tension induced by ethanol was inhibited when the strips were exposed to NG-monomethyl-L-arginine (L-NMMA). 6. Ethanol induced a gradual and sustained increase in [Ca2+]i in normal PSS, and a transient, concentration-dependent increase in [Ca2+]i in Ca(2+)-free PSS in porcine aortic valvular endothelial cells in situ.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of diazepam on the oxytocin induced contraction of the isolated rat uterus

This study was designed to investigate the effect of diazepam on the spontaneous contraction and oxytocin induced contraction of the isolated rat uterus. Female rat(Sprague-Dawley) pretreated with oophorectomy and 4 days administration of estrogen, weighing about 200 g, was sacrificed by cervical dislocation, and the uteruses were isolated. A longitudinal muscle strip was placed in temperature controlled() muscle chamber containing Locke's solution and myographied isometrically. Diazepam inhibited the spontaneous contraction and oxytocin induced contraction of the isolated rat uterus in a concentration-dependent manner. GABA, muscimol, a GABA A receptor agonist, bicuculline, a competitive GAGA A receptor antagonist, picrotoxin, a non competitive GABA A receptor antagonist, baclofen, a GABA B receptor agonist, and delta-aminovaleric acid, a GABA B receptor antagonist, did not affect on the spontaneous and oxytocin induced contraction of the isolated rat uterus. The inhibitory actions of diazepam on the spontaneous and oxytocin induced contraction were not affected by all the GABA receptor agonists and antagonists, but exceptionally potentiated by bicuculline. This potentiation-effect by bicuculline was not antagonized by muscimol. In normal calcium PSS, addition of calcium restored the spontaneous contraction preinhibited by diazepam and recovered the contractile of oxytocin preinhibited by diazepam. A23187, a calcium inophore, enhanced the restoration of both the spontaneous and oxytocin induced contraction by addition of calcium. In calcium-free PSS, diazepam suppressed the restoration of spontaneous motility by addition of calcium but allowed the recovery of spontaneous motility to a considerable extent. Diazepam could not inhibit some development of contractility by oxytocin in calcium-free PSS, but inhibited the increase in contractility by subsequent addition of calcium. These results suggest that the inhibitory action of diazepam on the rat uterine motility does not depend on or related to GABA receptors and that diazepam inhibits the extracellular calcium influx to suppress the spontaneous and oxytocin induced contractilities.

Effects of inositol phosphates on the membrane activity of smooth muscle cells of the rabbit portal vein.

The effects of intracellular perfusion of inositol 1,4,5-trisphosphate (InsP3) or inositol 1,3,4,5-tetrakisphosphate (InsP4) on electrical responses of smooth muscle cell membranes of the rabbit portal vein were studied using the whole cell voltage clamp technique. Depolarisation to 0 mV from a holding potential of -60 mV, evoked inward Ca (Ica), transient outward (ISO), oscillatory outward (IOO) and sustained outward (ISO) currents. Generation of IOO was dependent on the [Ca]o, but it was also generated in 0 mM Ca solution for over 10 min. From amplitude histograms, IOO was divided into two components. Reduction in [Ca]o inhibited the appearance of but not the amplitudes of both IOO components. However, the larger component of IOO was more resistant to a reduction in [Ca]o than the smaller one. InsP3 (10 microM) increased the frequency of both IOO components to a greater extent than their amplitude, but the larger component was more sensitive to InsP3 than the smaller one. The increase in the occurrence of IOO induced by InsP3 did not occur following pretreatment with 3 mM caffeine or 1 nM A23187. In normal PSS, InsP3 was evoked by a depolarising pulse positive to -40 mV, whereas following perfusion with InsP3 (10 microM), IOO was evoked at -60 mV. In normal PSS, intracellular perfusion with 10 microM InsP4 changed neither the frequency nor the amplitude of IOO, and the amplitudes of ICa, ITO and ISO were also unchanged. However, in 10 mM Ca solution, 10 microM InsP4 generated IOO at a membrane potential of -60 mV.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calcium withdrawal and agents affecting calcium translocation on agonist-induced contractions of the rabbit ovarian artery.

The effect of extracellular calcium ion (Ca2+) depletion, diltiazem, D-600 and TMB-8 on potassium (K+), norepinephrine-(NE) and histamine-induced contractions of the rabbit isolated ovarian artery was studied. K+-induced contractions, which were biphasic, were rapidly lost (congruent to 5 min) when calcium was removed from the physiological saline solution (O-Ca2+ PSS). NE and histamine-induced contractions also declined rapidly under nominal O-Ca2+ conditions without any evidence of a depletion resistant component. However, after 1 h under these conditions there was a partial restoration of their responses. EGTA (10(-3) mol/l) abolished these contractions to NE and histamine but not those in normal PSS. Diltiazem and D-600 were more effective against K+-induced than NE- and histamine-induced contractions while the intracellular Ca2+ antagonist TMB-8 was equally effective against all three responses. Histamine could still evoke contraction of the K+-contracted ovarian artery after this had been completely abolished with diltiazem or D-600 but not TMB-8. These results are interpreted to suggest that K+, NE and histamine probably release intracellular Ca2+ to evoke contractions of the ovarian artery. They differ, however, in the mechanisms they employ to facilitate entry of extracellular calcium, which in turn leads to intracellular calcium release.

Ion-selective electrode studies of cell Na components in vascular smooth muscle of WKY and SHR.

Vascular smooth muscle cells of the rat tail artery were enriched with Na by overnight incubation at 10 degrees C in K-free physiological salt solution (K-free PSS). The cells were then returned to normal PSS in a series of steps so as to define the effect of varying temperature between 3 and 37 degrees C, the effect of ouabain, the effect of readmitting K+ in the continuing presence of ouabain, and, finally, the effect of removing ouabain in the continuing presence of K+. The effects were measured with flow-through Na+ and K+ glass-cannula electrodes as changes in a superfusate moving slowly past the artery. Where possible, confirmatory evidence was obtained by conventional incubations followed by chemical analysis of the tissues. About 10 mmol Na+/kg dry wt is transferred out of the cells when temperature is raised from 3 to 37 degrees C. Exposure of the tissue to ouabain while the tissue is still in the K-free state produces a further loss of about 8 mmol Na+/kg dry wt and 4 mmol k+/kg dry wt. Readmittance of K+ in the continuing presence of ouabain induces a further loss of about 25 mmol Na+/kg dry wt in exchange for an uptake of about 13 mmol K+ measured electrometrically or about 20 mmol K measured chemically. Finally, the removal of ouabain is followed by an extrusion of about 155 mmol Na+/kg dry wt in exchange for an uptake of 133 mmol K%. The ouabain-insensitive exchange of Na+ and K+ is enhanced in spontaneously hypertensive rats as compared with normotensive rats.