Ectopic fat storage in heart, blood vessels and kidneys in the pathogenesis of cardiovascular diseases.

In humans and most animal models, the development of obesity leads not only to increased fat depots in classical adipose tissue locations but also to significant lipid deposits within and around other tissues and organs, a phenomenon known as ectopic fat storage. The purpose of this review is to explore the possible locations of ectopic fat in key target-organs of cardiovascular control (heart, blood vessels and kidneys) and to propose how ectopic fat storage can play a role in the pathogenesis of cardiovascular diseases associated with obesity. In animals fed a high-fat diet, cardiac fat depots within and around the heart impair both systolic and diastolic functions, and may in the long-term promote heart failure. Accumulation of fat around blood vessels (perivascular fat) may affect vascular function in a paracrine manner, as perivascular fat cells secrete vascular relaxing factors, proatherogenic cytokines and smooth muscle cell growth factors. Furthermore, high amounts of perivascular fat could mechanically contribute to the increased vascular stiffness seen in obesity. Finally, accumulation of fat in the renal sinus may limit the outflow of blood and lymph from the kidney, which would alter intrarenal physical forces and promote sodium reabsorption and arterial hypertension. Taken together, ectopic fat storage in key target-organs of cardiovascular control may impair their functions, contributing to the increased prevalence of cardiovascular diseases in obese subjects.

The function of Arg-94 in the oxidation and decarboxylation of glutaryl-CoA by human glutaryl-CoA dehydrogenase.

Glutaryl-CoA dehydrogenase catalyzes the oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and CO(2). Inherited defects in the protein cause glutaric acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydrogenase is the only member of the acyl-CoA dehydrogenase family with a cationic residue, Arg-94, situated in the binding site of the acyl moiety of the substrate. Crystallographic investigations suggest that Arg-94 is within hydrogen bonding distance of the gamma-carboxylate of glutaryl-CoA. Substitution of Arg-94 by glycine, a disease-causing mutation, and by glutamine, which is sterically more closely related to arginine, reduced k(cat) of the mutant dehydrogenases to 2-3% of k(cat) of the wild type enzyme. K(m) of these mutant dehydrogenases for glutaryl-CoA increases 10- to 16-fold. The steady-state kinetic constants of alternative substrates, hexanoyl-CoA and glutaramyl-CoA, which are not decarboxylated, are modestly affected by the mutations. The latter changes are probably due to steric and polar effects. The dissociation constants of the non-oxidizable substrate analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, are not altered by the mutations. However, abstraction of a alpha-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstraction of the alpha-proton of acetoacetyl-CoA by Arg-94 --> Gln mutant dehydrogenase is unaffected, and the resulting enolate forms a charge transfer complex with the oxidized flavin. These experiments indicate that Arg-94 does not make a major contribution to glutaryl-CoA binding. However, the electric field of Arg-94 may stabilize the dianions resulting from abstraction of the alpha-proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain gamma-carboxylates. It is also possible that Arg-94 may orient glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an alpha-proton.

The altered structure of renal papillary outflow tracts in obesity.

Weight gain is associated with an expanded renal medullary interstitium in humans and in animal models of obesity. In this study, the consequence of obesity and this expanded matrix on renal papillary structure was examined in 15 obese rabbits fed a high fat diet for 8-12 weeks compared to 21 rabbits fed a standard diet. When examined under a dissecting microscope, the tips of the renal papillae from formalin-fixed, methylene blue-stained kidneys showed patent ducts of Bellini in 5 of 5 instances from 2 lean rabbits, but in only 2 out of 12 ducts from 3 obese rabbits. The ostia of the remaining ducts were significantly distended (205 +/- 42 microns versus 56 +/- 8 microns) and occupied by lightly staining granular material. When examined with scanning electron microscopy, all ducts were patent in lean rabbits (6 ducts in 4 rabbits, averaging 104 +/- 12 microns across), whereas only 6 of 11 ducts were patent in papillae from 4 obese rabbits. Renal medullary parenchymal tissue appeared at the openings of the remaining 5 ducts of Bellini in the 4 rabbits. Not only were these 5 ducts significantly distended by the interstitial material (with openings averaging 248 +/- 56 microns across), but the associated collecting ducts were dilated relative to control (100 +/- 15 microns versus 75 +/- 7 microns). Since the ducts of Bellini are the only renal openings that are not corsetted by a fibrous capsule, the authors speculate that the expanded medullary interstitium and increased renal sinus lipid partially obstruct renal outflow and elevate renal interstitial hydrostatic pressure in obesity, causing a prolapse of parenchymal contents, further obstructing urine outflow and leading to distention of the collecting ducts and ducts of Bellini.

Proton abstraction reaction, steady-state kinetics, and oxidation-reduction potential of human glutaryl-CoA dehydrogenase.

Glutaryl-CoA dehydrogenase catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA and CO(2) in the mitochondrial degradation of lysine, hydroxylysine, and tryptophan. We have characterized the human enzyme that was expressed in Escherichia coli. Anaerobic reduction of the enzyme with sodium dithionite or substrate yields no detectable semiquinone; however, like other acyl-CoA dehydrogenases, the human enzyme stabilizes an anionic semiquinone upon reduction of the complex between the enzyme and 2,3-enoyl-CoA product. The flavin potential of the free enzyme determined by the xanthine-xanthine oxidase method is -0.132 V at pH 7.0, slightly more negative than that of related flavoprotein dehydrogenases. A single equivalent of substrate reduces 26% of the dehydrogenase flavin, suggesting that the redox equilibrium on the enzyme between substrate and product and oxidized and reduced flavin is not as favorable as that observed with other acyl-CoA dehydrogenases. This equilibrium is, however, similar to that observed in isovaleryl-CoA dehydrogenase. Comparison of steady-state kinetic constants of glutaryl-CoA dehydrogenase with glutaryl-CoA and the alternative substrates, pentanoyl-CoA and hexanoyl-CoA, suggests that the gamma-carboxyl group of glutaryl-CoA stabilizes the enzyme-substrate complex by at least 5.7 kJ/mol, perhaps by interaction with Arg94 or Ser98. Glu370 is positioned to function as the catalytic base, and previous studies indicate that the conjugate acid of Glu370 also protonates the transient crotonyl-CoA anion following decarboxylation [Gomes, B., Fendrich, G. , and Abeles, R. H. (1981) Biochemistry 20, 3154-3160]. Glu370Asp and Glu370Gln mutants of glutaryl-CoA dehydrogenase exhibit 7% and 0. 04% residual activity, respectively, with human electron-transfer flavoprotein; these mutations do not grossly affect the flavin redox potentials of the mutant enzymes. The reduced catalytic activities of these mutants can be attributed to reduced extent and rate of substrate deprotonation based on experiments with the nonoxidizable substrate analogue, 3-thiaglutaryl-CoA, and kinetic experiments. Determination of these fundamental properties of the human enzyme will serve as the basis for future studies of the decarboxylation reaction which is unique among the acyl-CoA dehydrogenases.

Distribution of renal medullary hyaluronan in lean and obese rabbits.

BACKGROUND: Obese individuals have an expanded interstitium in the renal inner medulla (IM), which stains positively with periodic acid-Schiff and Alcian blue. In obese dogs, the IM is also expanded, with hyaluronan (HA) content being 2.4 times control. METHODS: We determined the anatomic pattern of renal HA deposition following weight gain, using an animal model of obesity consisting of young rabbits (N = 10), representing animals entering into the study, lean rabbits (N = 19), fed a control diet, and obese rabbits (N = 19), fed a high-fat diet (15% fat, by fortifying with corn oil and lard, in a ratio of 2:1) for two to three months. Tissue was papain digested, and HA was recovered in a phosphate or a Tris buffer and detected by an indirect immunoabsorbent competition assay. RESULTS: Rabbits fed a high-fat diet for 8 to 12 weeks gained weight (37%) and became mildly hypertensive (10 mm Hg). In lean rabbits, HA was low in the renal cortex (6 +/- 30 microg/g tissue), increased steadily across the outer medulla (OM; 79 +/- 28 microg/g tissue) and was uniformly high in the IM (192 +/- 28 microg/g tissue) when recovered in a Tris buffer; these levels of tissue HA did not change during the three-month period of dietary intervention. In obese rabbits, the renal medullary interstitium was expanded and stained intensely with periodic acid Schiff and Alcian blue, and tissue HA was elevated in the IM (448 +/- 25 microg/g tissue) but not the cortex (5 +/- 25 microg/g tissue) or the OM (85 +/- 25 microg/g tissue). The significant difference was due to those IM samples taken from the renal papilla; IM samples from the body of the kidney did not significantly differ among the lean, obese, and young rabbits. CONCLUSION: The elevated renal HA associated with weight gain is limited to the IM and occurs most consistently in the papilla, which is the region of the kidney that is most vulnerable to distention caused by elevated renal interstitial hydrostatic pressure.

Human neutrophil elastase releases two pools of mucinlike glycoconjugate from tracheal submucosal gland cells.

Neutrophil elastase can contribute to the pathogenesis of increased airway reactivity and excess mucus secretion in many pulmonary diseases. Ten nanomolar human neutrophil elastase (HNE) effectively empties airway serous cells, raising the question of why HNE is not equally effective at emptying mucous cells of their stored mucin because total release of mucin granules is not seen in postmortem examination of even the most severe disease. To better resolve the mucus secretagogue action of HNE, we measured secretion of mucinlike glycoconjugates (MGCs) released from freshly isolated swine tracheal submucosal gland cells in fractions of the superfusate acquired every 2 min. Six to fifty nanomolar HNE released a fixed quantity of MGCs at an increasing rate with increasing concentrations of enzyme, an action consistent with the release of cell surface mucinlike molecules. The polycation poly-L-lysine (1 microg/ml) released a similar transient of MGCs. A steady-state doubling of MGC rate of release was seen as long as 100 nM HNE was present, but this stimulus represented less than a 1% release of stored MGCs/min and was consistent with release of mucin vesicles from cell stores. Both actions of HNE were inhibited by the specific inhibitors L-680833 and DMP-777 but not by 30 microM erythromycin. Therefore, HNE release of MGCs from tracheal submucosal glands is limited by both the fixed quantity of the MGCs in the transient pool and by the small steady-state response to the higher concentrations of enzyme.

The functions of the flavin contact residues, alphaArg249 and betaTyr16, in human electron transfer flavoprotein.

Arg249 in the large (alpha) subunit of human electron transfer flavoprotein (ETF) heterodimer is absolutely conserved throughout the ETF superfamily. The guanidinium group of alphaArg249 is within van der Waals contact distance and lies perpendicular to the xylene subnucleus of the flavin ring, near the region proposed to be involved in electron transfer with medium chain acyl-CoA dehydrogenase. The backbone amide hydrogen of alphaArg249 is within hydrogen bonding distance of the carbonyl oxygen at the flavin C(2). alphaArg249 may modulate the potentials of the two flavin redox couples by hydrogen bonding the carbonyl oxygen at C(2) and by providing delocalized positive charge to neutralize the anionic semiquinone and anionic hydroquinone of the flavin. The potentials of the oxidized/semiquinone and semiquinone/hydroquinone couples decrease in an alphaR249K mutant ETF generated by site directed mutagenesis and expression in Escherichia coli, without major alterations of the flavin environment as judged by spectral criteria. The steady state turnover of medium chain acyl-CoA dehydrogenase and glutaryl-CoA dehydrogenase decrease greater than 90% as a result of the alphaR249Ks mutation. In contrast, the steady state turnover of short chain acyl-CoA dehydrogenase was decreased about 38% when alphaR249K ETF was the electron acceptor. Stopped flow absorbance measurements of the oxidation of reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA product complex by wild type human ETF at 3 degrees C are biphasic (t(1/2)=12 ms and 122 ms). The rate of oxidation of this reduced binary complex of the dehydrogenase by the alphaR249K mutant ETF is extremely slow and could not be reasonably estimated. alphaAsp253 is proposed to function with alphaArg249 in the electron transfer pathway from medium chain acyl-CoA dehydrogenase to ETF. The steady state kinetic constants of the dehydrogenase were not altered when ETF containing an alphaD253A mutant was the substrate. However, t(1/2) of the rapid phase of oxidation of the reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA charge transfer complex almost doubled. betaTyr16 lies on a loop near the C(8) methyl group, and is also near the proposed site for interflavin electron transfer with medium chain acyl-CoA dehydrogenase. The tyrosine residue makes van der Waals contact with the C(8) methyl group of the flavin in human ETF and Paracoccus denitrificans ETF (as betaTyr13) and lies at a 30 degrees C angle with the plane of the flavin. Human betaTyr16 was substituted with leucine and alanine residues to investigate the role of this residue in the modulation of the flavin redox potentials and in electron transfer to ETF. In betaY16L ETF, the potentials of the flavin were slightly reduced, and steady state kinetic constants were modestly altered. Substitution of an alanine residue for betaTyr16 yields an ETF with potentials very similar to the wild type but with steady state kinetic properties similar to betaY16L ETF. It is unlikely that the beta methyl group of the alanine residue interacts with the flavin C(8) methyl. Neither substitution of betaTyr16 had a large effect on the fast phase of ETF reduction by medium chain acyl-CoA dehydrogenase.

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (

Renal size and composition in hypertensive, obese rabbits.

OBJECTIVE: To determine whether the renal growth associated with obesity is due to hypertrophy or hyperplasia. DESIGN: New Zealand white female rabbits were fed either standard rabbit chow (n=17) or chow fortified with 10% corn oil plus 5% lard (n=18) for 12-16 weeks. MEASUREMENTS: All rabbits were weighed, and intra-arterial blood pressures were successfully measured at the end of the study in 16 lean and 18 obese rabbits; percent water of entire kidneys (8 lean, 8 obese rabbits) and of defined regions of kidneys (8 lean, 10 obese rabbits) were obtained gravimetrically. Renal hemoglobin, protein and DNA was measured chemically (8 lean, 8 obese rabbits). RESULTS: Kidneys grew in size as the rabbits gained fat. In a series of 8 lean and 8 age-matched obese rabbits, weighing 3.7+/-0.1 kg and 5.4+/-0.4kg (P<0.05), the kidneys were 20% larger in the obese rabbits: 15.0+/-0.9 g vs 18.0+/-2.5 g (P<0.05). Kidney protein was also 20% greater in the obese rabbit: 1.38+/-0.06 g/kidney vs 1.66+/-0.06 g/kidney (P<0.05). While total renal DNA was 16% greater in the obese: 18.2+/-0.5 microg/kidney vs 21.1+/-0.61 g/kidney (P<0.05), no significant difference existed when the DNA was expressed as microg/mg protein. Fractional water content of the intact kidney declined with obesity: 78.7+/-1.1% vs 76.0+/-1.2% (P<0.05). Conversely, the hemoglobin content of the kidney at autopsy, an estimate of the unstressed vascular volume, increased with obesity: 55+/-19 mg/kidney vs 82+/-25 mg/kidney (P<0.05). By contrast, water content of renal parenchyma was constant: 80.8+/-1.0% vs 80.9+/-1.2% (cortex); 84.0+/-0.8% vs 83.6%+/-2.0% (outer medulla); and 85.7+/-0.8% vs 86.0+/-2.1% (inner medulla). CONCLUSION: The renal growth associated with obesity was predominantly hyperplastic and was associated with a partial exclusion of fluid from the renal sinus.

Reduction in the bioelectric properties of swine tracheal submucosal gland cells in culture after daily short-term exposure to cocaine.

Chronic use of cocaine has been associated with respiratory complications. In this study, we investigated the effects of daily short-term cocaine exposure on epithelial bioelectric properties and chloride secretion in response to secretagogues in primary culture of swine tracheal submucosal gland cells grown on microporous inserts. Cell cultures exposed continuously to cocaine for 24 h or intermittently for 30 min daily for up to 3 consecutive days, resulted in a concentration-dependent reduction in transwell voltage and transepithelial resistance. Cocaine (300 microM) treatment for 24 h decreased the voltage and resistance by 87 and 75%, respectively. The voltage and resistance were also substantially decreased after 3 days of intermittent cocaine (10-30 microM) exposure. Cocaine exposure protocols used here did not enhance lactate dehydrogenase (LDH) release. Chloride secretion was measured as short-circuit current utilizing Ussing chamber methodology. Cocaine exposure did not change the decreases in short-circuit current caused by amiloride (10 microM), but reduced the increases in short-circuit current induced by acetylcholine and isoproterenol. After 3 days of intermittent cocaine (30 microM) exposure, the maximal acetylcholine and isoproterenol responses were reduced by 67 and 71%, respectively. Therefore, cocaine exposure continuously for 24 h or intermittently for 30 min daily for up to 3 days decreased basal transepithelial voltage as well as resistance and reduced the responses to cholinergic and beta-adrenoceptor agonists. These results suggest that alterations in epithelial function can occur even after daily transient cocaine exposure.

Mucus glycoconjugate secretion in cool and hypertonic solutions.

For sensitive individuals, exercise-induced asthma is triggered by cold and dry air and is often accompanied by a productive cough. In this study, we determined whether cold solutions and/or solutions of increased tonicity directly caused an increase in glycoconjugate (GC) secretion. To test this, we used isolated swine tracheal submucosal gland cells (TSGCs) and measured the rate of GC secretion at 37 and 32 degrees C in isotonic solutions and in solutions made hypertonic by 30 mosM. TSGCs were isolated under conditions that minimized the rate of GC secretion and were perfused with medium 199 equilibrated with 5% CO2 to a pH of 7.4. A lectin-based assay was used to specifically detect GC present in each 2-min fraction of the perfusate. Basal secretion was 3.1-fold greater at 32 degrees C (n = 3) than at 37 degrees C (n = 4; P < 0.05). At 37 degrees C, increasing perfusate osmolarity by 30 mosM increased the average rate of secretion by 41 +/- 11% (n = 4; P < 0.05); return to isotonic perfusate caused a 4.5 +/- 1.8-fold transient increase in secretion (n = 4; P < 0.05) that was complete within 10 min. At 32 degrees C, changing tonicity of the perfusate had no significant effect but returning to isotonic perfusate caused a 2.3 +/- 0.7-fold transient increase in secretion (n = 3; P < 0.05). Thus key stimuli that trigger obstruction of airflow (cold and increased osmolarity) can also directly stimulate GC secretion in the airway. Such increased secretions may contribute to the productive cough observed in some individuals in response to cold air.

alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein.

Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.

Teaching physiology and the World Wide Web: electrochemistry and electrophysiology on the Internet.

Students seek active learning experiences that can rapidly impart relevant information in the most convenient way possible. Computer-assisted education can now use the resources of the World Wide Web to convey the important characteristics of events as elemental as the physical properties of osmotically active particles in the cell and as complex as the nerve action potential or the integrative behavior of the intact organism. We have designed laboratory exercises that introduce first-year medical students to membrane and action potentials, as well as the more complex example of integrative physiology, using the dynamic properties of computer simulations. Two specific examples are presented. The first presents the physical laws that apply to osmotic, chemical, and electrical gradients, leading to the development of the concept of membrane potentials; this module concludes with the simulation of the ability of the sodium-potassium pump to establish chemical gradients and maintain cell volume. The second module simulates the action potential according to the Hodgkin-Huxley model, illustrating the concepts of threshold, inactivation, refractory period, and accommodation. Students can access these resources during the scheduled laboratories or on their own time via our Web site on the Internet (http./(/)phys-main.umsmed.edu) by using the World Wide Web protocol. Accurate version control is possible because one valid, but easily edited, copy of the labs exists at the Web site. A common graphical interface is possible through the use of the Hypertext mark-up language. Platform independence is possible through the logical and arithmetic calculations inherent to graphical browsers and the Javascript computer language. The initial success of this program indicates that medical education can be very effective both by the use of accurate simulations and by the existence of a universally accessible Internet resource.

Effects of Elevated Cytosolic Calcium on ACh-Induced Swine Tracheal Smooth Muscle Contraction.

Increased intracellular calcium concentration ([Ca(2+)](i)) is required for smooth muscle contraction. In tracheal and other tonic smooth muscles, contraction and elevated [Ca(2+)](i) are maintained as long as an agonist is present. To evaluate the physiological role of steady-state increases in Ca(2+) on tension maintenance, [Ca(2+)](i) was elevated using ionomycin, a Ca(2+) ionophore or charybdotoxin, a large-conductance calcium-activated potassium channel (K(Ca)) blocker prior to or during exposure of tracheal smooth muscle strips to ACh (10(-9) to 10(-4) M). Ionomycin (5 &mgr;M) in resting muscles induced increases in [Ca(2+)](i) to 500 +/- 230 nM and small increases in force of 2.6 +/- 2.3 N/cm(2). This tension is only 10% of the maximal tension induced by ACh. Charybdotoxin had no effect on [Ca(2+)](i) or tension in resting muscle. After pretreatment of muscle with ionomycin, the concentration-response relationship for ACh-induced changes in tension shifted to the left (EC(50) = 0.07 +/- 0.05 &mgr;M ionomycin; 0.17 +/- 0.07 &mgr;M, control, p < 0.05). When applied to the muscles during steady-state responses to submaximal concentrations of ACh, both ionomycin and charybdotoxin induced further increases in tension. The same magnitude increase in tension occurs after ionomycin and charybdotoxin treatment, even though the increase in [Ca(2+)](i) induced by charybdotoxin is much smaller than that induced by ionomycin. We conclude that the resting muscle is much less sensitive to elevation of [Ca(2+)](i) when compared to muscles stimulated with ACh. Steady-state [Ca(2+)](i) limits tension development induced by submaximal concentrations of ACh. The activity of K(Ca) moderates the response of the muscle to ACh at concentrations less than 1 &mgr;M. Copyright 1996 S. Karger AG, Basel

Cocaine inhibits chloride secretion by swine tracheal submucosal gland cells grown in culture.

Active ion transport by airway submucosal gland cells plays an important role in maintaining effective mucociliary clearance. Alterations in the clearance will affect normal airway functions. Nasal and airway mucosal surfaces are exposed to high concentrations of cocaine after snorting crystals or smoking the freebase form, causing respiratory complications. The actions of cocaine on airway gland cell ion transport were determined by measuring the basal electrical properties and ion transport activated by acetylcholine (ACh) and isoproterenol (ISO) in swine tracheal submucosal gland cells grown in culture. The cells were enzymatically isolated, purified on Percoll density gradients, and then grown on microporous inserts at an air interface. Chloride secretion was measured as short-circuit current (Isc) utilizing Ussing chamber methodology. Amiloride (10 microM) was added mucosally to block sodium reabsorption. The maximal changes in Isc (delta Isc) induced by ACh and ISO were 42.0 +/- 4.4 and 8.8 +/- 0.8 microA/cm2. Cocaine decreased basal Isc and the increase in Isc induced by ACh and ISO. The concentration-response relationships for ACh and ISO were shifted to the right by cocaine (1-3 mM) when applied serosally. The maximal increase in Isc induced by ISO was inhibited by serosal cocaine, but, the maximal response to ACh was not. Thus, cocaine inhibits ACh-induced increases in Isc by competitively blocking the receptor and the response to ISO by noncompetitive inhibition. Mucosal cocaine reduced the maximal response to ACh and ISO and also caused a rightward shift. We conclude that cocaine directly alters submucosal gland cell function by reducing the basal and stimulated ability to transport ions and fluid.

Hypertension, cardiac hypertrophy, and neurohumoral activity in a new animal model of obesity.

Although obesity is a major risk factor for morbidity and mortality, the mechanisms mediating cardiovascular abnormalities in response to weight gain are unclear. One reason for the paucity of information in this area is the lack of appropriate animal models for the study of human obesity. Therefore, the goal of the present study was to develop a small animal model of dietary-induced obesity that mimics many of the characteristics of human obesity. We studied female New Zealand White rabbits fed either a normal (n = 17) or high-fat diet (n = 15) and examined the cardiovascular consequences of obesity, including changes in blood pressure, humoral activation, and end-organ effects such as cardiac hypertrophy. After 12 wk, rabbits on the high-fat diet were 46% heavier than their lean counterparts (5.49 +/- 0.09 vs. 3.77 +/- 0.06 kg, respectively; P = 0.0001). Obese rabbits had higher resting heart rates than lean rabbits (220 +/- 7 vs. 177 +/- 6 beats/min; P = 0.0001) and developed hypertension (96 +/- 2 vs. 85 +/- 1 mmHg; P = 0.0001), hyperinsulinemia (32.5 +/- 3.4 vs. 15.5 +/- 1.0 microU/ml; P = 0.0001), hyperglycemia (162.4 +/- 2.9 vs. 141.9 +/- 2.7 mg/dl; P = 0.0001), and elevated triglycerides (102.3 +/- 9.1 vs. 48.5 +/- 4.0 mg/dl; P = 0.0001). Obese rabbits also developed cardiac hypertrophy, as evidenced by left ventricular (LV) dry weights that were 52% greater in obese than in lean rabbits (P = 0.0003). In addition, LV total protein was increased in proportion to the increase in LV weight. The results of this study suggest that rabbits fed a high-fat diet for a period of 12 wk develop many of the characteristics of human obesity. The obese rabbit should provide a small and relatively inexpensive animal model to investigate mechanisms of obesity-related cardiovascular abnormalities.

Renal sinus lipomatosis and body composition in hypertensive, obese rabbits.

OBJECTIVE: To test whether renal lipomatosis, an accretion of fat in the renal sinus associated with chronic renal infections, abscesses and calculi, can also be caused by rapid weight gain. DESIGN: New Zealand white rabbits were fed either standard rabbit chow (n = 24) or chow fortified with 10% corn oil plus 5% lard (n = 25) for 8-12 weeks. MEASUREMENTS: The rabbits and constituent tissues were weighed initially, after drying and after organic extractions. Renal tissue cholesterol and triglycerides were measured chemically. RESULTS: Rabbits made obese by increased fat intake were 1.8 kg heavier than controls (5.5 +/- 0.3 kg vs 3.7 +/- 0.2; n = 24,25), had 1.54 kg more body fat (1.90 +/- 0.25 vs 0.36 +/- 0.11 kg/rabbit; n = 10,9), and had a mean arterial blood pressure that was 9.2 mm Hg greater than controls (95.1 +/- 8.5 vs 85.9 +/- 5.6 mm Hg; n = 23,24). Individual organs grew in mass (lung, 15%; gastrocnemius, 17%; liver, 27%; kidney, 30%) and their parenchyma gained extractable lipids (lung, 5.5 mg/g tissue; gastrocnemius, 9.6 mg/g tissue; liver, 17.9 mg/g tissue). Total renal triglycerides were increased 2.1 fold, from 103 +/- 36 to 219 +/- 59 mg/kidney (n = 8,8), compared to the 5.3 fold increase in whole body fat. Renal cholesterol was increased 1.7 fold, from 7.5 +/- 1.1 to 12.7 +/- 2.9 mg/kidney, (n = 8,8). Within experimental error, the sum of the total renal triglycerides plus the total renal cholesterol equaled the net fat extracted from the renal sinus alone: 95 +/- 29 mg/kidney in lean rabbits and 253 +/- 71 mg/kidney in obese (n = 17,17). CONCLUSION: Obesity alone can cause renal lipomatosis. This increased volume of anatomically localized fat may be sufficient to externally compress renal veins and lymphatics, thus altering renal hemodynamic behavior.

Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue.

Syntrophin, a 58-kDa membrane-associated protein, is one component of a protein complex associated with dystrophin and other members of the dystrophin family, including the 87-kDa homologue (87K protein). To characterize interactions between synthrophin and 87K protein, we used an in vitro overlay binding assay. We demonstrate that purified Torpedo syntrophin binds directly to dystrophin and 87K. By expressing overlapping regions of the 87K protein as bacterial fusion proteins for binding targets, we show that a 52-amino acid region of 87K (residues 375-426) is sufficient for binding syntrophin.

Mouse alpha 1- and beta 2-syntrophin gene structure, chromosome localization, and homology with a discs large domain.

The syntrophin family of dystrophin-associated proteins consists of three isoforms, alpha 1, beta 1, and beta 2, each encoded by a distinct gene. We have cloned and characterized the mouse alpha 1- and beta 2-syntrophin genes. The mouse alpha 1-syntrophin gene ( > 24 kilobases) is comprised of eight exons. The mouse beta 2-syntrophin gene ( > 33 kilobases) contains seven exons, all of which have homologues at the corresponding position in the alpha 1-syntrophin gene. Primer extension analysis reveals two transcription initiation sites in the alpha 1-syntrophin gene and a single site in the beta 2-syntrophin gene. The sequence immediately 5' of the transcription start sites of both genes lacks a TATA box but is GC-rich and has multiple putative SP1 binding sites. The alpha 1-syntrophin gene is located on human chromosome 20 and mouse chromosome 2, while the beta 2-syntrophin gene is on human chromosome 16 and mouse chromosome 8. Analysis of the amino acid sequence of the syntrophins reveals the presence of four conserved domains. The carboxyl-terminal 56 amino acids are highly conserved and constitute a syntrophin unique domain. Two pleckstrin homology domains are located at the amino-terminal end of the protein. The first pleckstrin homology domain is interrupted by a domain homologous to repeated sequences originally found in the Drosophila discs-large protein.

Calcium Mobilization and Muscle Contraction Induced by Acetylcholine in Swine Trachealis.

The roles of Ca(2+) mobilization in development of tension induced by acetylcholine (ACh, 0.1-100 &mgr;M) in swine tracheal smooth muscle strips were studied. Under control conditions, ACh induced a transient increase in free cytosolic calcium concentration ([Ca(2+)](i)) that declined to a steady-state level. The peak increase in [Ca(2+)](i) correlated with the magnitude of tension at each [ACh] after a single exposure to ACh, while the steady-state [Ca(2+)](i) did not. Removal of extracellular Ca(2+) had little effect on peak [Ca(2+)](i) but greatly reduced steady-state increases in [Ca(2+)](i) and tension. Verapamil inhibited steady-state [Ca(2+)](i) only at [ACh] <1 &mgr;M. After depletion of internal Ca(2+) stores by 10 min exposure to ACh in Ca(2+)-free solution and then washout of ACh for 5 min in Ca(2+)-free solution, simultaneous re-exposure to ACh in the presence of 2.5 mM Ca(2+) increased [Ca(2+)](i) to the control steady-state level without overshoot. The tension attained was the same as control for each [ACh] used. Continuous exposure to successively increasing [ACh] (0.1-100 &mgr;M) also reduced the overshoot of [Ca(2+)](i) at 10 and 100 &mgr;M ACh, yet tension reached control levels at each [ACh] used. We conclude that the steady-state increase in [Ca(2+)](i) is necessary for tension maintenance and is dependent on Ca(2+) influx through voltage-gated calcium channels at 0.1 &mgr;M ACh and through a verapamil-intensitive pathway at 10 and 100 &mgr;M. The initial transient increase in calcium arises from intracellular stores and is correlated with the magnitude of tension only in muscles that have completely recovered from previous exposure to agonists. Copyright 2001 S. Karger AG, Basel