Biomedical subjects

R J Paul

Publications and source records attributed to R J Paul.

At least 19 recordsLinked to original sources

PubMed2002

Circulatory oxygen transport in the water flea Daphnia magna.

To determine the contribution of circulatory convection to tissue oxygen supply in animals of Daphnia magna, heart rate ( f(H)), in-vivo Hb oxygen-saturation ( S(Hb)) and NADH fluorescence intensity ( I(NADH)) as a measure of the tissue oxygenation state were simultaneously measured using digital motion analysis, microabsorption spectroscopy and fluorescence microscopy. In addition, the relationship between stroke volume and body size was established. Groups of differently sized animals (small: 1.4-1.6 mm, medium: 2.7-2.9 mm, large: 3.3 mm) with either low (Hb-poor) or high Hb concentration (Hb-rich) in the hemolymph were exposed to a gradual decrease in ambient oxygen partial pressure ( P(O2amb)) between normoxia and anoxia. In all groups, f(H) increased in response to progressive hypoxia. The hypoxic maximum in f(H) was highest in medium-sized Hb-poor animals, whereas perfusion rate increased continuously with increasing body size in Hb-poor and Hb-rich animals. The P(O2amb) at which Hb in the heart region was half-saturated (in-vivo P(50)) was higher in medium-sized (Hb-poor: 3.2 kPa, Hb-rich: 2.0 kPa) than in small (Hb-poor: 2.1 kPa, Hb-rich: 1.5 kPa) and large animals (Hb-poor: 1.9 kPa). The in-vivo P(50) was always lower in Hb-rich than in Hb-poor animals. The I(NADH) indicated an impairment of tissue oxygenation starting at higher critical P(O2amb) with increasing body size and with lower Hb concentration. Model calculations suggest that at the respective critical P(O2amb), circulatory convection delivers less than half of the oxygen demand in Hb-poor animals. In contrast, in Hb-rich animals, the contribution of circulatory convection to tissue oxygen supply at respective critical P(O2amb) was much greater due to the higher concentration of Hb.

Animals↗

PubMed2002

The sarcoplasmic reticulum and smooth muscle function: evidence from transgenic mice.

Smooth muscle Ca2+ handling is of major importance to understanding its function. A new approach utilizes molecular biology to develop transgenic mouse models in which the protein constituents of the various Ca2+ regulatory subsystems have been altered. Gene-targeted or gene-ablated (knockout) mice have been reported for the sarcoplasmic reticulum (SR) Ca2+ pump isoforms SERCA2, SERCA2a and SERCA3, the plasma membrane Ca2+ pump isoforms, PMCA1, PMCA2 and PMCA4, and the SR-associated protein, phospholamban (PLB), an inhibitor of SERCA2. A mouse line carrying a transgene for the smooth muscle specific expression of PLB has been reported. Evidence from studies using these mice combined with the classical pharmacological approaches has provided new insight into the relative role of the SR. We review this field with particular emphasis on PLB, since its modulation of SR function and smooth muscle contractility has the largest database. PLB via modulation of SERCA can play a major role in regulation of both phasic and tonic smooth muscle contractility. The use of transgenic mice has yielded surprises ,uch as PLB modulation of endothelial cell Ca2+ homeostasis, and the demonstration that PLB is the major site for A-kinase-mediated relaxation of mouse bladder. The use of these gene-altered models has provided evidence clearly implicating a major role for the SR in modulating smooth muscle Ca2+ and contractility, with the caveat that this modulation is tissue specific.

Animals↗

PubMed2002

Polylysine binding to unphosphorylated smooth muscle myosin enhances formation and stabilizes myosin filaments in vitro.

Previously, we demonstrated that positively charged polylysine, our model for biological polyamines, activates the Mg2+ ATPase activity of unphosphorylated smooth muscle myosin and shifts the myosin conformation from the folded 10S to linear 6S form. These effects of polylysine were reversed by the oppositely charged heparin (Szymanski et al. (1993) Am J Physiol 265, C379). In the present report, we provide further information on polylysine binding to smooth muscle myosin, and test the hypothesis that polylysine binding to unphosphorylated myosin involves filament formation. To relate the effects of polylysine on contractility in smooth muscle to physiologically relevant material, we investigated the ability of naturally occurring positively charged polyamines, histones, cadaverine, putrescine and spermidine to activate the Mg2+ ATPase activity of unphosphorylated smooth muscle myosin. Our data show that polylysine binding to individual unphosphorylated myosin molecules stimulates formation of myosin filaments. Polylysine also interacts with myosin filaments, causing enhancement of their size and the numbers, and this could be reversed by heparin. Polylysine binding to myosin filaments made them more resistant to disassembly by high salt concentrations (KCl) or ATP. Naturally occurring polyamines in millimolar concentrations activate the Mg2+ ATPase activity of unphosphorylated smooth muscle myosin. We suggest that the electrostatic interactions between naturally occurring positively charged polyamines and unphosphorylated smooth muscle myosin may play a role in stabilization of thick filament structurein situ.

Animals↗

PubMed2001

Aquaporin 5-deficient mouse lungs are hyperresponsive to cholinergic stimulation.

Although aquaporin 5 (AQP5) is the major water channel expressed in alveolar type I cells in the lung, its actual role in the lung is a matter of considerable speculation. By using immunohistochemical staining, we show that AQP5 expression in mouse lung is not restricted to type I cells, but is also detected in alveolar type II cells, and in tracheal and bronchial epithelium. Aqp5 knockout (Aqp5(-/-)) mice were used to analyze AQP5 function in pulmonary physiology. Compared with Aqp5(+/+) mice, Aqp5(-/-) mice show a significantly increased concentration-dependent bronchoconstriction to intravenously administered Ach, as shown by an increase in total lung resistance and a decrease in dynamic lung compliance (P < 0.05). Likewise, Penh, a measure of bronchoconstriction, was significantly enhanced in Aqp5(-/-) mice challenged with aerosolized methacholine (P < 0.05). The hyperreactivity to bronchoconstriction observed in the Aqp5(-/-) mice was not due to differences in tracheal smooth muscle contractility in isolated preparations or to altered levels of surfactant protein B. These data suggest a novel pathway by which AQP5 influences bronchoconstriction. This observation is of special interest because studies to identify genetic loci involved in airway hyperresponsiveness associated with asthma bracket genetic intervals on human chromosome 12q and mouse chromosome 15, which contain the Aqp5 gene.

Acetylcholine↗

PubMed2001

Phospholamban regulation of bladder contractility: evidence from gene-altered mouse models.

1. Phospholamban (PLB) is an inhibitor of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). Its presence and/or functional significance in contractility of bladder, a smooth muscle tissue particularly dependent on SR function, is unknown. We investigated this by measuring the effects of carbachol (CCh) on force and [Ca2+]i in bladder from mice in which the PLB gene was ablated (PLB-KO mice). In the PLB-KO bladder, the maximum increases in [Ca2+]i and force were significantly decreased (41.5 and 47.4 % of WT), and the EC50 values increased. 2. Inhibition of SERCA with cyclopiazonic acid (CPA) abolished these differences between WT and PLB-KO bladder, localizing the effects to the SR. 3. To determine whether these effects were specific to PLB, we generated mice with smooth-muscle-specific expression of PLB (PLB-SMOE mice), using the SMP8 alpha-actin promoter. Western blot analysis of PLB-SMOE mice showed approximately an eightfold overexpression of PLB while SERCA was downregulated 12-fold. 4. In PLB-SMOE bladders, in contrast, the response of [Ca2+]i and force to CCh was significantly increased and the EC50 values were decreased. CPA had little affect on the CCh-induced increases in [Ca2+]i and force in PLB-SMOE bladder. 5. These results show that alteration of the PLB:SERCA ratio can significantly modulate smooth muscle [Ca2+]i. Importantly, our data show that PLB can play a major role in modulation of bladder contractility.

Animals↗

PubMed2001

Is myosin phosphatase regulated in vivo by inhibitor-1? Evidence from inhibitor-1 knockout mice.

1. The Ca(2+) sensitivity of smooth muscle contractility is modulated via regulation of phosphatase activity. Protein phosphatase inhibitor-1 (I-1) is the classic type-1 phosphatase inhibitor, but its presence and role in cAMP-dependent protein kinase (PKA) modulation of smooth muscle is unclear. To address the relevance of I-1 in vivo, we investigated smooth muscle function in a mouse model lacking the I-1 protein (I-1((-/-)) mice). 2. Significant amounts of I-1 protein were detected in the wild-type (WT) mouse aorta and could be phosphorylated by PKA, as indicated by (32)P-labelled aortic extracts from WT mice. 3. Despite the significant presence of I-1 in WT aorta, phenylephrine and KCl concentration- isometric force relations in the presence or absence of the PKA pathway activator isoproterenol (isoprenaline) were unchanged compared to I-1((-/-)) aorta. cGMP-dependent protein kinase (PKG) relaxation pathways were also not different. Consistent with these findings, dephosphorylation rates of the 20 kDa myosin light chains (MLC(20)), measured in aortic extracts, were nearly identical between WT and I-1((-/-)) mice. 4. In the portal vein, I-1 protein ablation was associated with a significant (P < 0.05) rightward shift in the EC(50) of isoproterenol relaxation (EC(50) = 10.4 +/- 1.4 nM) compared to the WT value (EC(50) = 3.5 +/- 0.2 nM). Contraction in response to acetylcholine as well as Ca(2+) sensitivity were similar between WT and I-1((-/-)) aorta. 5. Despite the prevalence of I-1 and its activation by PKA in the aorta, I-1 does not appear to play a significant role in contractile or relaxant responses to any pharmacomechanical or electromechanical agonists used. I-1 may play a role as a fine-tuning mechanism involved in regulating portal vein responsiveness to beta-adrenergic agonists.

Acetylcholine↗

PubMed2001

Distinct pathways of Ca(2+) sensitization in porcine coronary artery: effects of Rho-related kinase and protein kinase C inhibition on force and intracellular Ca(2+).

Alterations of the Ca(2+) sensitivity of contraction have been reported for porcine coronary artery, but the mechanisms are not clearly understood. We investigated the mechanism(s) of Ca(2+) sensitization in response to the thromboxane A(2) analogue (U46619). Our hypothesis is that different mechanisms of Ca(2+) sensitization could be distinguished by their distinct time courses. Therefore, we measured the time course of [Ca(2+)](i) and isometric force simultaneously in an intact artery after a single addition of U46619. The initial transient phase was associated with Ca(2+) release from the sarcoplasmic reticulum, whereas the maintained phase was associated with Ca(2+) influx. Two distinct types of Ca(2+) sensitization characterized these phases with either protein kinase C (PKC)-mediated or Rho-kinase-mediated mechanisms. Their effects were quite distinct on the basis of the time courses over which the sensitization was effective. PKC inhibition (1 micromol/L calphostin C) had a much greater effect in the initial phase, diminishing the size of the transient and prolonging the rise in force and the decline in [Ca(2+)](i). There were limited effects on the sustained force. Rho-kinase inhibition (10 micromol/L Y27632), in contrast, nearly abolished the sustained force but had a lesser effect on the transient phase. Neither inhibitor had any effect on the force versus [Ca(2+)](i) relations for KCl contractures. Our evidence suggests that both PKC-mediated and Rho-kinase-mediated Ca(2+) sensitizations are present in coronary arteries, but the latter is dominant in thromboxane A(2) receptor-mediated contraction.

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

PubMed2001

Loss of SM-B myosin affects muscle shortening velocity and maximal force development.

We used an exon-specific gene-targeting strategy to generate a mouse model deficient only in the SM-B myosin isoform. Here we show that deletion of exon-5B (specific for SM-B) in the gene for the heavy chain of smooth muscle myosin results in a complete loss of SM-B myosin and switching of splicing to the SM-A isoform, without affecting SM1 and SM2 myosin content. Loss of SM-B myosin does not affect survival or cause any overt smooth muscle pathology. Physiological analysis reveals that absence of SM-B myosin results in a significant decrease in maximal force generation and velocity of shortening in smooth muscle tissues. This is the first in vivo study to demonstrate a functional role for the SM-B myosin isoform. We conclude that the extra seven-residue insert in the surface loop 1 of SM-B myosin is a critical determinant of crossbridge cycling and velocity of shortening.

Animals↗

PubMed2001

Dantrolene reduces serum TNFalpha and corticosterone levels and muscle calcium, calpain gene expression, and protein breakdown in septic rats.

The effects of dantrolene on serum TNFalpha and corticosterone levels and on muscle calcium, calpain gene expression, and protein breakdown were studied in rats with abdominal sepsis induced by cecal ligation and puncture. Treatment of rats with 10 mg/kg of dantrolene 2 h before and 8 h after induction of sepsis reduced serum TNFalpha and corticosterone, muscle calcium levels, mRNA levels for m- and mu-calpain, and the muscle specific calpain p94, as well as total and myofibrillar protein breakdown rates, determined as release of tyrosine and 3-methylhistidine, respectively, from incubated extensor digitorum longus muscles. The results support the concept that increased calcium concentrations may be an important mechanism of sepsis-induced muscle protein breakdown. The data also indicate that other mechanisms, in addition to reduced muscle calcium concentrations such as decreased levels of TNFalpha and glucocorticoids, may contribute to the anti-catabolic effects of dantrolene during sepsis. The observations are important from a clinical standpoint because they suggest that the catabolic response in skeletal muscle during sepsis may be prevented by treatment with a calcium antagonist.

Animals↗

PubMed2001

Hypoxic vasodilation in porcine coronary artery is preferentially inhibited by organ culture.

Hypoxia (95% N2-5% CO2) elicits an endothelium-independent relaxation (45-80%) in freshly dissected porcine coronary arteries. Paired artery rings cultured at 37 degrees C in sterile DMEM (pH approximately 7.4) for 24 h contracted normally to KCl or 1 microM U-46619. However, relaxation in response to hypoxia was sharply attenuated compared with control (fresh arteries or those stored at 4 degrees C for 24 h). Hypoxic vasorelaxation in organ cultured vessels was reduced at both high and low stimulation, indicating that both Ca2+-independent and Ca2+-dependent components are altered. In contrast, relaxation to G-kinase (sodium nitroprusside) or A-kinase (forskolin and isoproterenol) activation was not significantly affected by organ culture. Additionally, there was no difference in relaxation after washout of the stimulus, indicating that the inhibition is specific to acute hypoxia-induced relaxation. Simultaneous force and intracellular calcium concentration ([Ca2+]i) measurements indicate the reduction in [Ca2+]i concomitant with hypoxia at low stimulus levels in these tissue is abolished by culture. Our results indicate that organ culture at 37 degrees C specifically attenuates hypoxic relaxation in vascular smooth muscle by altering dynamics of [Ca2+]i handling and decreasing a Ca2+-independent component of relaxation. Thus organ culture can be a novel tool for investigating the mechanisms of hypoxia-induced vasodilation.

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

PubMed2001

Contractile regulation of the Na(+)-K(+)-2Cl(-) cotransporter in vascular smooth muscle.

Vasoconstrictors activate the Na(+)-K(+)-2Cl(-) cotransporter NKCC1 in rat aortic smooth muscle, but the mechanism is unknown. Efflux of (86)Rb(+) from rat aorta in response to phenylephrine (PE) was measured in the absence and presence of bumetanide, a specific inhibitor of NKCC1. Removal of extracellular Ca(2+) completely abolished the activation of NKCC1 by PE. This was not due to inhibition of Ca(2+)-dependent K(+) channels since blocking these channels with Ba(2+) in Ca(2+)-replete solution did not prevent activation of NKCC1 by PE. Stimulation of NKCC1 by PE was inhibited 70% by 75 microM ML-9, 97% by 2 microM wortmannin, and 70% by 2 mM 2,3-butanedione monoxime, each of which inhibited isometric force generation in aortic rings. Bumetanide-insensitive Rb(+) efflux, an indication of Ca(2+)-dependent K(+) channel activity, was reduced by ML-9 but not by the other inhibitors. Stretching of aortic rings on tubing to increase lumen diameter to 120% of normal almost completely blocked the stimulation of NKCC1 by PE without inhibiting the stimulation by hypertonic shrinkage. We conclude that activation of the Na(+)-K(+)-2Cl(-) cotransporter by PE is the direct result of smooth muscle contraction through Ca(2+)-dependent activation of myosin light chain kinase. This indicates that the Na(+)-K(+)-2Cl(-) cotransporter is regulated by the contractile state of vascular smooth muscle.

Androstadienes↗

PubMed2001

Chloride channel function is linked to epithelium-dependent airway relaxation.

We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial Cl- secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on Cl- channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the Cl- channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing Cl- in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for Cl- secretion using Cl- free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that Cl- channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive Cl- channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive Cl- channel other than CFTR is active in the SP response. This work introduces a possible role for Cl- pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies of airway function as well as therapeutic approaches to pulmonary disease.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2001

EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2.

Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2 from airway epithelium in other species, suggesting PGE2 as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2 receptor [EP2(-/-)], one of the PGE2 receptors, we tested the hypothesis that PGE2 is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(-/-) mice. There were no differences between EP2(-/-) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(-/-) and wild-type tracheas in basal or stimulated PGE2 production. Exogenous PGE2 produced significantly less relaxation in EP2(-/-) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2 receptors are of primary importance in airway relaxation to PGE2 and relaxation to SP or ATP is mediated through PGE2 acting on EP2 receptors.

Acetylcholine↗

PubMed2001

The alpha(1)- and alpha(2)-isoforms of Na-K-ATPase play different roles in skeletal muscle contractility.

The Na-K-ATPase, which maintains the Na(+) and K(+) gradients across the plasma membrane, can play a major role in modulation of skeletal muscle contractility. Although both alpha(1)- and alpha(2)-isoforms of the Na-K-ATPase are expressed in skeletal muscle, the physiological significance of these isoforms in contractility is not known. Evaluation of the contractile parameters of mouse extensor digitorum longus (EDL) was carried out using gene-targeted mice lacking one copy of either the alpha(1)- or alpha(2)-isoform gene of the Na-K-ATPase. The EDL muscles from heterozygous mice contain approximately one-half of the alpha(1)- or alpha(2)-isoform, respectively, which permits differentiation of the functional roles of these isoforms. EDL from the alpha(1)(+/-) mouse shows lower force compared with wild type, whereas that from the alpha(2)(+/-) mouse shows greater force. The different functional roles of these two isoforms are further demonstrated because inhibition of the alpha(2)-isoform with ouabain increases contractility of alpha(1)(+/-) EDL. These results demonstrate that the Na-K-ATPase alpha(1)- and alpha(2)-isoforms may play different roles in skeletal muscle contraction.

Animals↗

PubMed2001

Smooth muscle-targeted overexpression of insulin-like growth factor I results in enhanced vascular contractility.

Insulin-like growth factor I (IGF-I) has been postulated to function as a vasodilator. We explored the vasoactive effects of chronic elevations of arterial IGF-I levels in SMP8-IGF-I mice, in which IGF-I is overexpressed in smooth muscle (SM) by means of a SM alpha-actin promoter. Denuded aortas from SMP8-IGF-I mice generated increased force in response to KCl or phenylephrine and had greater sensitivity to KCl depolarization. This is not due to desensitization of a SM NO pathway, as pretreatment with n-omega-nitro-L-arginine affected both wild-type and SMP8-IGF-I aortas to a similar degree. The increased contractility ex vivo is not associated with changes in heart rate or blood pressure. Total smooth muscle myosin heavy chain (SMHC) messenger RNA (mRNA) was greater in SMP8-IGF-I aortas, with preferential expression of SMHC-A. Reciprocal effects on contractility and SMHC mRNA were observed in SMP8-IGFBP-4 animals, in which IGF-binding protein-4 was overexpressed through the same promoter. Also, SM alpha-actin mRNA was increased in the aortas from SMP8-IGF-I mice. In summary, chronic arterial overexpression of IGF-I is associated with increased contractility. These effects differ from those seen after acute exposure to the growth factor and may relate to IGF-mediated changes in expression and relative isoform abundance of critical contractile proteins.

Animals↗

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↗