Search PubMed⌕ Search

Biomedical subjects

R J Paul

Publications and source records attributed to R J Paul.

At least 37 records · Page 2Linked to original sources
PubMed2001

Benefits of haemoglobin in the cladoceran crustacean Daphnia magna.

To determine the contribution of haemoglobin (Hb) to the hypoxia-tolerance of Daphnia magna, we exposed Hb-poor and Hb-rich individuals (2.4-2.8 mm long) to a stepwise decrease in ambient oxygen partial pressure (P(O(2)amb)) over a period of 51 min from normoxia (20.56 kPa) to anoxia (<0.27 kPa) and looked for differences in their physiological performance. The haem-based concentrations of Hb in the haemolymph were 49 micromol l(-1) in Hb-poor and 337 micromol l(-1) in Hb-rich animals, respectively. The experimental apparatus made simultaneous measurement of appendage beating rate (fA), NADH fluorescence intensity (I(NADH)) of the appendage muscles, heart rate (fH) and in vivo Hb oxygen-saturation possible. In response to progressive, moderate hypoxia, both groups showed pronounced tachycardia and a slight decrease in fA. The fA and fH of Hb-rich animals were generally 4-6 % lower than those of Hb-poor animals. In addition, Hb-rich animals showed a significant decrease in the P(O(2)amb) at which the Hb in the heart region was half-saturated and a striking reduction in the critical P(O(2)amb) of appendage-related variables. In Hb-poor animals, the I(NADH) signal indicated that the oxygen supply to the limb muscle tissue started to become impeded at a critical P(O(2)amb) of 4.75 kPa, although the high level of fA was largely maintained until 1.77 kPa. The obvious discrepancy between these two critical P(O(2)amb) values suggested an anaerobic supplementation of energy provision in the range 4.75-1.77 kPa. The fact that I(NADH) of Hb-rich animals did not rise until P(O(2)amb) fell below 1.32 kPa strongly suggests that the extra Hb available to Hb-rich animals ensured an adequate oxygen supply to the limb muscle tissue in the P(O(2)amb) range 4.75-1.32 kPa. This finding illustrates the physiological benefit of Hb in enabling the animal to sustain its aerobic metabolism as the energetically most efficient mode of fuel utilization under conditions of reduced oxygen availability.

Animals↗
PubMed2000

Preferential role of intracellular Ca2+ stores in regulation of isometric force in NIH 3T3 fibroblast fibres.

Fibroblast contraction plays a major role in wound repair, but the regulatory mechanisms are not well known. We investigated the relations between isometric force and intracellular calcium concentration ([Ca2+]i) in fibroblast fibres. These fibres were made with mouse NIH 3T3 fibroblasts cultured with native collagen in a three-dimensional matrix. Calf serum (CS; 30%) elicited a monotonic increase in force that attained a maximum within 15 min and could be sustained indefinitely. In contrast, [Ca2+]i increased to a peak at 3 min after CS stimulation, then returned to baseline levels by 10 min. Pretreatment with Ca2+-free medium or the Ca2+-channel antagonist nicardipine (10 microM) blocked the CS-induced [Ca2+]i increase, but force was not affected. KCl (50 mM) stimulation on the other hand, elicited a prolonged increase in [Ca2+]i but did not increase force. Inhibition of the endoplasmic reticulum Ca2+ release with Ca2+-ATPase inhibitors cyclopiazonic acid (5 microM) or thapsigargin (5 microM) nearly abolished (<20% control) the increase in [Ca2+]i and force response to CS. Treatment with ryanodine (10 microM) and caffeine (20 mM) had a similar effect. The phospholipase C inhibitor U73122 (3 microM) reduced the CS-induced increases in [Ca2+]i and force by 70 and 40%, respectively. We conclude that fibroblast isometric force is not coupled to Ca2+ arising from transmembrane influx but is correlated with the transient [Ca2+]i increase due to release from intracellular stores. Store-released Ca2+ may initiate activation pathways for fibroblast force development, but is not required for force maintenance.

3T3 Cells↗
PubMed2000

Effects of hypoxia on isometric force, intracellular Ca(2+), pH, and energetics in porcine coronary artery.

When exposed to hypoxic conditions, coronary arteries dilate, which is an important protective response. Although vessel sensitivity to oxygen is well documented, the mechanisms are not known with certainty. To further characterize the mechanisms of oxygen sensing in the coronary artery, we tested the major classes of hypotheses by measuring the effects of hypoxia on energetics, [Ca(2+)](i), K(+) channel function, and pH(i). Hypoxia relaxes porcine coronary arteries stimulated with either KCl or U46619. The extent of relaxation is dependent on both the degree and kind of stimulation. [Ca(2+)](i) was measured in endothelium-denuded arteries using fura 2-AM and ratiometric fluorescent techniques. At lower stimulus levels, hypoxia decreased both force and [Ca(2+)](i). Inhibitor studies suggest that K(Ca) and K(ATP) channels are not involved in the hypoxic relaxation, whereas K(V) channels may play a minor role, if any. Despite the hypoxia-mediated decrease in force, [Ca(2+)](i) was unchanged or increased at high levels of stimulation. Despite a marked increase in lactate content, pH(i) (measured with the ratiometric fluorescent dye BCECF) was also little affected by hypoxia. Measurement of the phosphagen and metabolite profile of freeze-clamped arteries with analytical isotachophoresis indicated that hypoxia increased lactate content by 4-fold and decreased phosphocreatine to 60% of control. However, neither ATP nor P(i) was affected by hypoxia. Interestingly, additional stimulation under hypoxia increased force but not ATP utilization, as estimated from measurements of anaerobic lactate production. Thus, surprisingly, the economy of force maintenance is increased under hypoxia. In porcine coronary artery, both Ca(2+)-dependent and, importantly, Ca(2+)-independent mechanisms are involved in hypoxic vasodilatation. For the latter, mechanisms involving either ATP, [Ca(2+)](i), pH(i), or P(i) cannot be invoked. This novel oxygen sensing mechanism involves a decreased Ca(2+) sensitivity.

Animals↗
PubMed2000

Metabolic adaptations to environmental changes in Caenorhabditis elegans.

Metabolic adaptations to environmental changes were studied in Caenorhabditis elegans. To assess adjustments in enzyme function, maximum activities of key enzymes of main metabolic pathways were determined. After a 12 h incubation at varying temperatures (10, 20 degrees C) and oxygen supplies (normoxia or anoxia), the activities of the following enzymes were determined at two measuring temperatures in tissue extracts: lactate dehydrogenase (LDH; anaerobic glycolysis), 3-hydroxyacyl-CoA-dehydrogenase (HCDH; fatty acid oxidation), isocitrate dehydrogenases (NAD-IDH, NADP-IDH; tricarboxylic acid cycle) and isocitrate lyase (ICL; glyoxylate cycle). Incubation at 20 degrees C induced a strong increase in maximum LDH activity. Anoxic incubation caused maximum HCDH and NADP-IDH activities and, at 10 degrees C incubation, LDH activity to increase. Maximum NAD-IDH and ICL activities were not influenced by any type of incubation. In order to study the time course of metabolic adaptations to varying oxygen supplies, relative quantities of free and protein-bound NADH were determined in living C. elegans using time-resolved fluorescence spectroscopy. During several hours of anoxia, free and protein-bound NADH showed different time courses. One main result was that just at the moment when the protein-bound NADH had reached a constant level, and the free NADH started to increase rapidly, the worms fell into a rigor state. The data on enzyme activity and NADH fluorescence can be interpreted on the basis of a two-stage model of anaerobiosis.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

A role for smad6 in development and homeostasis of the cardiovascular system.

Smad proteins are intracellular mediators of signalling initiated by Tgf-betasuperfamily ligands (Tgf-betas, activins and bone morphogenetic proteins (Bmps)). Smads 1, 2, 3, 5 and 8 are activated upon phosphorylation by specific type I receptors, and associate with the common partner Smad4 to trigger transcriptional responses. The inhibitory Smads (6 and 7) are transcriptionally induced in cultured cells treated with Tgf-beta superfamily ligands, and downregulate signalling in in vitro assays. Gene disruption in mice has begun to reveal specific developmental and physiological functions of the signal-transducing Smads. Here we explore the role of an inhibitory Smad in vivo by targeted mutation of Madh6 (which encodes the Smad6 protein). Targeted insertion of a LacZ reporter demonstrated that Smad6 expression is largely restricted to the heart and blood vessels, and that Madh6 mutants have multiple cardiovascular abnormalities. Hyperplasia of the cardiac valves and outflow tract septation defects indicate a function for Smad6 in the regulation of endocardial cushion transformation. The role of Smad6 in the homeostasis of the adult cardiovascular system is indicated by the development of aortic ossification and elevated blood pressure in viable mutants. These defects highlight the importance of Smad6 in the tissue-specific modulation of Tgf-beta superfamily signalling pathways in vivo.

Animals↗
PubMed2000

Effects of microtubules and microfilaments on [Ca(2+)](i) and contractility in a reconstituted fibroblast fiber.

We used a reconstituted fiber formed when 3T3 fibroblasts are grown in collagen to characterize nonmuscle contractility and Ca(2+) signaling. Calf serum (CS) and thrombin elicited reversible contractures repeatable for >8 h. CS elicited dose-dependent increases in isometric force; 30% produced the largest forces of 106 +/- 12 microN (n = 30), which is estimated to be 0.5 mN/mm(2) cell cross-sectional area. Half times for contraction and relaxation were 4.7 +/- 0.3 and 3.1 +/- 0.3 min at 37 degrees C. With imposition of constant shortening velocities, force declined with time, yielding time-dependent force-velocity relations. Forces at 5 s fit the hyperbolic Hill equation; maximum velocity (V(max)) was 0.035 +/- 0. 002 L(o)/s. Compliance averaged 0.0076 +/- 0.0006 L(o)/F(o). Disruption of microtubules with nocodazole in a CS-contracted fiber had no net effects on force, V(max), or stiffness; force increased in 8, but decreased in 13, fibers. Nocodazole did not affect baseline intracellular Ca(2+) concentration ([Ca(2+)](i)) but reduced ( approximately 30%) the [Ca(2+)](i) response to CS. The force after nocodazole treatment was the primary determinant of stiffness and V(max), suggesting that microtubules were not a major component of fiber internal mechanical resistance. Cytochalasin D had major inhibitory effects on all contractile parameters measured but little effect on [Ca(2+)](i).

3T3 Cells↗
PubMed2000

H(2)O(2) mediates Ca(2+)- and MLC(20) phosphorylation-independent contraction in intact and permeabilized vascular muscle.

One purpose of the current study was to establish whether vasoconstriction occurs in all vessel types in response to H(2)O(2). Isometric force was measured in pulmonary venous and arterial rings, and isobaric contractions were measured in mesenteric arteries and veins in response to H(2)O(2). A second purpose was to determine whether H(2)O(2)-induced contraction is calcium independent. The addition of H(2)O(2) to calcium-depleted (using the Ca(2+) ionophore ionomycin in zero calcium EGTA buffer) muscle caused contraction. Furthermore, permeabilized muscle contracted in response to H(2)O(2) even in zero Ca(2+). The final purpose was to determine whether the 20-kDa regulatory myosin light chain (MLC(20)) phosphorylation plays a role in H(2)O(2)-induced contraction. Pulmonary arterial strips were freeze-clamped at various time points during H(2)O(2)-induced contractions, and the relative amounts of phosphorylated MLC(20) were measured. H(2)O(2) caused dose-dependent contractions that were independent of MLC(20) phosphorylation. ML-9, a myosin light chain kinase inhibitor, had no effect on the H(2)O(2) contractile response. In conclusion, H(2)O(2) induces Ca(2+)- and MLC(20) phosphorylation-independent contraction in pulmonary and systemic arterial and venous smooth muscle.

Animals↗
PubMed2000

Effects of microtubule disruption on force, velocity, stiffness and [Ca(2+)](i) in porcine coronary arteries.

Force generated by smooth muscle cells is believed to result from the interaction of actin and myosin filaments and is regulated through phosphorylation of the myosin regulatory light chain (LC(20)). The role of other cytoskeleton filaments, such as microtubules and intermediate filaments, in determining the mechanical output of smooth muscle is unclear. In cultured fibroblasts, microtubule disruption results in large increases in force similar to contractions associated with LC(20) phosphorylation (15). One hypothesis, the "tensegrity" or "push-pull" model, attributes this increase in force to the disruption of microtubules functioning as rigid struts to resist force generated by actin-myosin interaction (9). In porcine coronary arteries, the disruption of microtubules by nocodazole (11 microM) also elicited moderate but significant increases in isometric force (10-40% of a KCl contracture), which could be blocked or reversed by taxol (a microtubule stabilizer). We tested whether this nocodazole-induced force was accompanied by changes in coronary artery stiffness or unloaded shortening velocity, parameters likely to be highly sensitive to microtubule resistance elements. Few changes were seen, ruling out push-pull mechanisms for the increase in force by nocodazole. In contrast, the intracellular calcium concentration, measured by fura 2 in the intact artery, was increased by nocodazole in parallel with force, and this was inhibited and/or reversed by taxol. Our results indicate that microtubules do not significantly contribute to vascular smooth muscle mechanical characteristics but, importantly, may play a role in modulation of Ca(2+) signal transduction.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1999

Transgenic overexpression of beta(2)-adrenergic receptors in airway smooth muscle alters myocyte function and ablates bronchial hyperreactivity.

beta(2)-Adrenergic receptors (beta(2)AR) act to relax airway smooth muscle and can serve to counteract hyperresponsiveness, although the effect may not be ablative even in the presence of exogenous agonist. Within this signaling cascade that ultimately transduces smooth muscle relaxation, a significant "spare receptor" pool has been hypothesized to be present in the airway. In order to modify the relationship between beta(2)AR and downstream effectors, transgenic mice (TG) were created overexpressing beta(2)AR approximately 75-fold in airway smooth muscle using a mouse smooth muscle alpha-actin promoter. While >90% of these receptors were expressed on the smooth muscle cell surface, the percentage of receptors able to form the agonist-promoted high affinity complex was less than that found with nontransgenic (NTG) cells (R(H) = 18 versus 36%). Nevertheless, beta(2)AR signaling was found to be enhanced. Intact airway smooth muscle cells from TG had basal cAMP levels that were greater than NTG cells. A marked increase in agonist-stimulated cAMP levels was found in the TG ( approximately 200% stimulation over basal) compared with NTG ( approximately 50% over basal) cells. Adenylyl cyclase studies gave similar results and also showed a 10-fold lower EC(50) for TG cells. Tracheal rings from TG mice that were precontracted with acetylcholine had an enhanced responsiveness (relaxation) to beta-agonist, with a 60-fold decrease in the ED(50), indicating that the enhanced signaling imposed by overexpression results in an increase in the coordinated function of the intact airway cells. In vivo studies showed a significantly blunted airway resistance response to the inhaled bronchoconstrictor methacholine in the TG mice. Indeed, with beta-agonist pretreatment, the TG mice displayed no response whatsoever to methacholine. These results are consistent with beta(2)AR being the limiting factor in the transduction system. Increases in the initial component of this transduction system (the beta(2)AR) are sufficient to markedly alter signaling and airway smooth muscle function to the extent that bronchial hyperresponsiveness is ablated, consistent with an anti-asthma phenotype.

Adenylyl Cyclases↗
PubMed1999

Phospholamban is present in endothelial cells and modulates endothelium-dependent relaxation. Evidence from phospholamban gene-ablated mice.

Vascular endothelial cells regulate vascular smooth muscle tone through Ca2+-dependent production and release of vasoactive molecules. Phospholamban (PLB) is a 24- to 27-kDa phosphoprotein that modulates activity of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Expression of PLB is reportedly limited to cardiac, slow-twitch skeletal and smooth muscle in which PLB is an important regulator of [Ca2+]i and contractility in these muscles. In the present study, we report the existence of PLB in the vascular endothelium, a nonmuscle tissue, and provide functional data on PLB regulation of vascular contractility through its actions in the endothelium. Endothelium-dependent relaxation to acetylcholine was attenuated in aorta of PLB-deficient (PLB-KO) mice compared with wild-type (WT) controls. This effect was not due to actions of nitric oxide on the smooth muscle, because sodium nitroprusside-mediated relaxation in either denuded or endothelium-intact aortas was unaffected by PLB ablation. Relative to denuded vessels, relaxation to forskolin was enhanced in WT endothelium-intact aortas. The endothelium-dependent component of this relaxation was attenuated in PLB-KO aortas. To investigate whether these changes were due to PLB, WT mouse aorta endothelial cells were isolated. Both reverse transcriptase-polymerase chain reaction and Western blot analyses revealed the presence of PLB in endothelial cells, which were shown to be >98% pure by diI-acetylated LDL uptake and nuclear counterstaining. These data indicate that PLB is present and modulates vascular function as a result of its actions in endothelial cells. The presence of PLB in endothelial cells opens new fields for investigation of Ca2+ regulatory pathways in nonmuscle cells and for modulation of endothelial-vascular interactions.

Animals↗
PubMed1999

Impaired cardiac performance in heterozygous mice with a null mutation in the sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene.

The sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2 (SERCA2) gene encodes both SERCA2a, the cardiac sarcoplasmic reticulum Ca2+ pump, and SERCA2b, which is expressed in all tissues. To gain a better understanding of the physiological functions of SERCA2, we used gene targeting to develop a mouse in which the promoter and 5' end of the gene were eliminated. Mating of heterozygous mutant mice yielded wild-type and heterozygous offspring; homozygous mutants were not observed. RNase protection, Western blotting, and biochemical analysis of heart samples showed that SERCA2 mRNA was reduced by approximately 45% in heterozygous mutant hearts and that SERCA2 protein and maximal velocity of Ca2+ uptake into the sarcoplasmic reticulum were reduced by approximately 35%. Measurements of cardiovascular performance via transducers in the left ventricle and right femoral artery of the anesthetized mouse revealed reductions in mean arterial pressure, systolic ventricular pressure, and the absolute values of both positive and negative dP/dt in heterozygous mutants. These results demonstrate that two functional copies of the SERCA2 gene are required to maintain normal levels of SERCA2 mRNA, protein, and Ca2+ sequestering activity, and that the deficit in Ca2+ sequestering activity due to the loss of one copy of the SERCA2 gene impairs cardiac contractility and relaxation.

Animals↗
PubMed1999

Epipodite and fat cells as sites of hemoglobin synthesis in the branchiopod crustacean Daphnia magna.

In contrast to the malacostracan crustaceans that use hemocyanin as the oxygen carrier, a number of branchiopod crustaceans, such as the water flea Daphnia magna, utilize hemoglobin (Hb) as the respiratory protein. By means of in situ hybridization (ISH) techniques with subsequent signal amplification using catalyzed reporter deposition, sites of Hb synthesis were localized in Daphnia magna. Based on a previously reported Hb-cDNA sequence, a specific ISH probe was designed and hybridized with the Hb-mRNA in histological sections of adult D. magna. The detection of Hb-mRNA was tissue specific and revealed that Hb is synthesized in fat cells, which play a role in fat and glycogen metabolism, and in epithelial cells of the epipodites, which are involved in osmoregulation. Sites of Hb synthesis have been identified in several invertebrate phyla, including Annelida and Nematoda. However, this is the first example in the class Crustacea, and only the second in the phylum Arthropoda.

Adipocytes↗
PubMed1999

Anaerobiosis in the nematode Caenorhabditis elegans.

In Caenorhabditis elegans, mortality rates and changes in concentrations of carbohydrate stores and anaerobic end products were determined in anoxic (test) and normoxic (control) animals at two different temperatures (10 and 20 degrees C). The anoxic tolerance of the free-living nematode proved to be well-developed: at 10 degrees C, about 50% of animals had survived a period of 50 h of anoxia. The carbohydrate stores (approximately 30 mmol glycosyl units kg-1 freshweight (FW)) were reduced by two-thirds within 24 h of anoxia at both temperatures. L-lactate, acetate, succinate, and propionate were identified as the main anaerobic end products. The amounts and proportions of the end products were dependent on temperature. They did not accumulate very much in the tissues, but were mainly excreted. During anoxia, the metabolism of C. elegans was depressed to 3-4% of the aerobic value. The food-source Escherichia coli was found to be at least partly alive in the gut of the animals. To separate between anaerobiosis in animals and bacteria, cleaning procedures were applied, and additional control measurements were made: anaerobic end products produced either by E. coli alone or by bacteria-free (axenic) bred nematodes were quantified at identical incubation conditions.

Acetates↗
PubMed1999

Vasoconstrictors and nitrovasodilators reciprocally regulate the Na+-K+-2Cl- cotransporter in rat aorta.

Little is known about the function and regulation of the Na+-K+-2Cl- cotransporter NKCC1 in vascular smooth muscle. The activity of NKCC1 was measured as the bumetanide-sensitive efflux of 86Rb+ from intact smooth muscle of the rat aorta. Hypertonic shrinkage (440 mosmol/kgH2O) rapidly doubled cotransporter activity, consistent with its volume-regulatory function. NKCC1 was also acutely activated by the vasoconstrictors ANG II (52%), phenylephrine (50%), endothelin (53%), and 30 mM KCl (54%). Both nitric oxide and nitroprusside inhibited basal NKCC1 activity (39 and 34%, respectively), and nitroprusside completely reversed the stimulation by phenylephrine. The phosphorylation of NKCC1 was increased by hypertonic shrinkage, phenylephrine, and KCl and was reduced by nitroprusside. The inhibition of NKCC1 significantly reduced the contraction of rat aorta induced by phenylephrine (63% at 10 nM, 26% at 30 nM) but not by KCl. We conclude that the Na+-K+-2Cl- cotransporter in vascular smooth muscle is reciprocally regulated by vasoconstrictors and nitrovasodilators and contributes to smooth muscle contraction, indicating that alterations in NKCC1 could influence vascular smooth muscle tone in vivo.

Angiotensin II↗
PubMed1999

Stimulation of both aerobic glycolysis and Na(+)-K(+)-ATPase activity in skeletal muscle by epinephrine or amylin.

Epinephrine and amylin stimulate glycogenolysis, glycolysis, and Na(+)-K(+)-ATPase activity in skeletal muscle. However, it is not known whether these hormones stimulate glycolytic ATP production that is specifically coupled to ATP consumption by the Na(+)-K(+) pump. These studies correlated glycolysis with Na(+)-K(+)-ATPase activity in resting rat extensor digitorum longus and soleus muscles incubated at 30 degrees C in well-oxygenated medium. Lactate production rose three- to fourfold, and the intracellular Na(+)-to-K(+) ratio (Na(+)/K(+)) fell with increasing concentrations of epinephrine or amylin. In muscles exposed to epinephrine at high concentrations (5 x 10(-7) and 5 x 10(-6) M), ouabain significantly inhibited glycolysis by approximately 70% in either muscle and inhibited glycogenolysis by approximately 40 and approximately 75% in extensor digitorum longus and soleus, respectively. In the absence of ouabain, but not in its presence, statistically significant inverse correlations were observed between lactate production and intracellular Na(+)/K(+) for each hormone. Epinephrine had no significant effect on oxygen consumption or ATP content in either muscle. These results suggest for the first time that stimulation of glycolysis and glycogenolysis in resting skeletal muscle by epinephrine or amylin is closely linked to stimulation of active Na(+)-K(+) transport.

Adenosine Triphosphate↗
PubMed1999

[Ca2+]i homeostasis and cyclic nucleotide relaxation in aorta of phospholamban-deficient mice.

Phospholamban (PLB), a protein localized in the sarcoplasmic reticulum (SR), inhibits the SR Ca2+-ATPase; phosphorylation of PLB relieves this inhibition. We previously reported significant differences in contractility in aorta from mice in which the gene for PLB was ablated (PLB-). In this study, we measured intracellular Ca2+ concentration ([Ca2+]i) with fura 2 in the intact mouse aorta to more directly test the hypothesis that these changes are ascribable to altered SR function in vivo. Ten micromoles per liter of the alpha-agonist phenylephrine (PE) increased [Ca2+]i monotonically to a steady state in the wild-type aorta. In contrast, in PLB- aorta there was an initial rapid increase to a peak [Ca2+]i, which then decreased to a steady state that was lower than that in the wild type. Upon removal of the stimulus (either PE or KCl), the decrease in [Ca2+]i was two times as fast in the PLB- as in the wild-type aorta. There were no significant differences between PLB- and wild-type aortas in the concentration vs. force relations or the time courses of relaxation in response to forskolin or sodium nitroprusside. Interestingly, stimulation of the cAMP pathway before cGMP pathway activation resulted in a significant increase in sensitivity and a difference in relaxation parameters between PLB- and wild-type aortas. Western blot analysis indicated that the PLB-to-sarcoendoplasmic reticulum Ca2+ATPase ratio in the mouse aorta was similar to that in the heart; 20-fold more aortic than heart homogenate was required to achieve a similar level of immunoreactivity. Our data indicate that PLB can play a major role in modulating smooth muscle [Ca(2+)](i) but only a minor role, if any, in cyclic nucleotide-mediated relaxation.

Animals↗
PubMed1999

Ablation of the SERCA3 gene alters epithelium-dependent relaxation in mouse tracheal smooth muscle.

Sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3), an isoform of the intracellular Ca(2+) pump that has been shown to mediate endothelium-dependent relaxation of vascular smooth muscle, is also expressed in tracheal epithelium. To determine its possible role in regulation of airway mechanical function, we compared tracheal contractility in gene-targeted mice deficient in SERCA3 (SERCA3(-)) with that in wild-type tracheae. Cumulative addition of ACh elicited concentration-dependent increases in isometric force (ED(50) = 2 microM, maximum force = 8 mN/mm(2)) that were identical in SERCA3(-) and wild-type tracheae. After ACh stimulation, substance P (SP) elicited a transient relaxation (42.6 +/- 3.2%, n = 28) in both tracheae. However, the rate of relaxation was significantly (P < 0.04, n = 9) more rapid in the wild-type [half-time (t(1/2)) = 34.3 s] than in the SERCA3(-) (t(1/2) = 61.6 s) trachea. The SP relaxation was reduced by rubbing the trachea, indicative of epithelial cell involvement. This was verified using a perfused trachea preparation. SP in the outside medium had no effect, whereas SP in the perfusate bathing the epithelial side elicited a relaxation. Nitric oxide synthase inhibition (0.2 mM N(omega)-nitro-L-arginine) reduced the SP relaxation by 36.5 +/- 12.5%, whereas the SP effect was abolished by eicosanoid inhibition (10 microM indomethacin). ATP also elicited an epithelium-dependent relaxation similar to SP but with a more rapid relaxation in the SERCA3(-) trachea than in the wild-type trachea. Our results indicate that SERCA3 gene ablation does not directly affect smooth muscle, which is consistent with the distribution of the isoform, but suggest that SERCA3 plays a role in epithelial cell modulation of airway smooth muscle function.

Acetylcholine↗